Enzymatic esterification of (-)-menthol with fatty acids in solvent by a commercial lipase from Candida rugosa

Article abstract of DOI:10.1007/s11746-001-0219-x

Esterification of (-)-menthol with fatty acids in isooctane was successfully catalyzed using a commercial lipase, Lipase AY 'Amano' 30 from Candida rugosa in original powder form. The esterification reactions were performed to elucidate the effects of temperature, enzyme load, molar ratio of (-)-menthol/fatty acid, and fatty acid type, keeping the (-)-menthol concentration at 200 mM. At the optimal conditions for (-)-menthol esterification, determined at a (-)-menthol/lauric acid molar ratio of 1:1 and 35°C [1.5 g enzyme/g (-)-menthol, 0.1 g molecular sieves], the molar conversion of (-)-menthol after 48 h reached 93%. After 24 h, the lowest and the highest molar conversions of fatty acids at 2:1 molar ratio were obtained with myristic acid (71%) and margaric acid (98%), respectively. After 48 h, the molar conversions of lauric acid at molar ratios 2:1, 1:1, and 1:2 were 98, 93, and 49%, respectively.

Full text of DOI:10.1007/s11746-001-0219-x

Enzymatic Esterification of (−)-Menthol with Fatty Acids
in Solvent by a Commercial Lipase from Candida rugosa
Banu Babali, H. Ays¸e Aksoy*, Melek Tüter, and Guldem Ustun
Istanbul Technical University, Chemical Engineering Department, 80626, Maslak, Istanbul, Turkey
ABSTRACT: Esterification of (−)-menthol with fatty acids in
isooctane was successfully catalyzed using a commercial lipase,
Lipase AY “Amano” 30 from Candida rugosa in original powder
form. The esterification reactions were performed to elucidate
the effects of temperature, enzyme load, molar ratio of
(−)-menthol/fatty acid, and fatty acid type, keeping the (−)-men-
thol concentration at 200 mM. At the optimal conditions for
(−)-menthol esterification, determined at a (−)-menthol/lauric acid
molar ratio of 1:1 and 35°C [1.5 g enzyme/g (−)-menthol, 0.1 g
molecular sieves], the molar conversion of (−)-menthol after 48 h
reached 93%. After 24 h, the lowest and the highest molar con-
versions of fatty acids at 2:1 molar ratio were obtained with myris-
tic acid (71%) and margaric acid (98%), respectively. After 48 h,
the molar conversions of lauric acid at molar ratios 2:1, 1:1, and
1:2 were 98, 93, and 49%, respectively.
ification of (−)-menthol. Kamiya et al. (4,5) investigated the
enantioselective esterification of menthol with saturated and
unsaturated long-chain fatty acids in solvent. Surfactant (non-
ionic, glutamic acid dioleyl ester ribitol amide)-coated lipase
from C. cylindracea was found to be highly selective for
the esterification of (−)-menthol. The rate of esterification of
(−)-menthol with lauric acid catalyzed by surfactant-coated li-
pase was more than 100 times that of the powder lipase. There-
fore, (−)-menthol esterification using the surfactant-coated li-
pase from C. cylindracea (also termed C. rugosa, commercial
name: Lipase AY produced by Amano Pharmaceutical Co.)
was investigated to elucidate the effects of temperature, kind
of solvent, and n-saturated fatty acids with different chain
lengths. The optimal temperature was around 35°C. The cat-
alytic activity and enantioselectivity of the lipase were found
to depend strongly on the type of organic solvents. Isooctane
was found to be the best solvent, giving high initial rates of re-
action and enantioselectivity (4).
Paper no. J9590 in JAOCS 78, 53–56 (January 2001).
KEY WORDS: Candida cylindracea lipase, Candida rugosa li-
pase, enzymatic esterification, fatty acid, (−)-menthol.
The initial rate of (−)-menthol esterification increased with
Menthol (p-menthan-3-ol) is a secondary terpene alcohol; it has increasing fatty acid chain length. Kamiya and Goto (5) con-
eight optically active isomers that have different organoleptic cluded that the enzymatic esterification of menthol with lauric
properties. (−)-Menthol has a characteristic peppermint flavor acid using surfactant-coated lipase from C. rugosa (Lipase AY)
and refreshing coolness. The other isomers of menthol do not conformed to the ping-pong bi-bi mechanism in which the re-
have this cooling effect (1). Because of its flavor and refresh- action was inhibited by excess menthol. A lipase from C. ru-
ing coolness, (−)-menthol is widely used in foods, cosmetics, gosa (Lipase AY-30) catalyzed the enatioselective resolution
and pharmaceutics. (−)-Menthol is produced from natural of racemic menthol using acid anhydrides as acylating agents
sources such as peppermint oils of Mentha piperita and M. ar- (6). Thus, stereoselective resolution of racemic menthol with
venisis. Their free-menthol contents range between 70 and vinyl propionate as the acylating agent was catalyzed in organic
80%. It is also obtained industrially by the optical resolution of solvents by an immobilized lipase from Pseudomonas cepacia
the racemic mixture produced by organic synthesis, which is (PS-30). In reactions conducted in hexane at a 1:1 mole ratio
an equal molar mixture of (+)- and (−)-menthol (1). The reso- of racemic menthol to vinyl propionate, the mole percentage
lution of racemic mixtures can be carried out by esterification, yield of (−)-menthyl propionate increased from 50 to 86%
column chromatography, or crystallization.
when the reaction temperature was increased from 20 to 60°C.
Enzymatic esterification is a highly selective method com- The highest increase in yield was obtained between 40 and
pared with other resolution methods. Enzymatic esterification 50°C. However, increasing the temperature decreased the stereo-
of (−)-menthol and enzymatic resolution of racemic menthol specificity toward (−)-menthol. It is well known that the polar-
have been investigated with lipases of different origins in a ity of organic solvents affects the activity of biocatalyst. The
water–oil emulsion (2,3) or in solvent (4–6) to elucidate the ef- polarity of organic solvents can be measured by the value of
fects of temperature, solvents, and fatty acid chain lengths. Li- log P (the logarithm of the partition coefficient of a given sol-
pases (EC 3.1.1.3) have been widely used for the resolution of vent between water and 1-octanol). Among the investigated
racemic alcohols through enantiospecific esterification and solvents, cylohexane (log P = 3.2), toluene (log P = 2.5), and
transesterification. Among the several lipases investigated, li- benzene (log P = 2.0) gave low yields of (−)-menthol propi-
pases originating from Candida cylindracea and Penicillium onate but with a good enantiomeric ratio and enantiomeric ex-
simplicissimum were found to be highly selective for the ester- cess while solvents of log P values above 3.5 such as isooctane
(log P = 4.5), n- heptane (log P = 4.0), and n-hexane (log P =
3.5) gave high yields and stereoselectivity (7).
*To whom correspondence should be addressed.
E-mail: aksoyha@itu.edu.tr
Copyright © 2001 by AOCS Press
53
JAOCS, Vol. 78, no. 1 (2001)

54
B. BABALI ET AL.
90°C for 15 min to inactivate the enzyme and then centrifuged
(−)-Menthol was esterified with long-chain unsaturated fatty
to separate the ester product. The ester products were dried with
anhydrous Na2SO4 and analyzed by capillary gas chromatogra-
phy (see next paragraph). By using the number of micromoles
of menthyl esters produced after 1 min per gram of enzyme, the
initial rates of the reaction were calculated.
acids (oleic, linoleic, and α-linolenic acids) in an organic sol-
vent-free system using commercial lipase from C. rugosa (8).
The optimal conditions were determined as follows: (−)-men-
thol/fatty acid molar ratio of 1:3, 30% water, 700 units en-
zyme/g reaction mixture, 30°C. After 24 h, the esterification
yields of (−)-menthol with oleic, linoleic, and α-linolenic acids
were 96, 88, and 95%, respectively. The reaction showed high
enantioselectivity. After 32 h, the enantiomeric ratio and enan-
tiomeric excess of (−)-menthol were 31 and 88%, respectively.
In this study we used lipase powder from C. rugosa (Lipase
AY “Amano” 30), without coating with a surfactant, as a cata-
lyst to investigate the enzymatic esterification of (−)-menthol
with saturated fatty acids of even and odd numbers of carbon
atoms (lauric, myristic, palmitic, margaric, and stearic) and un-
saturated (oleic) fatty acid in isooctane. Furthermore, we eval-
uated the effects of the temperature, type of fatty acid, (−)-men-
thol/fatty acid molar ratio, and enzyme content of the reaction
mixture on the esterification reaction.
Analysis of the esterification product. The ester products,
which were mixtures of menthyl esters, unreacted (−)-menthol,
fatty acids and isooctane, were analyzed using an HP 5890 Se-
ries II gas chromatograph (Hewlett-Packard, Waldron, Ger-
many) equipped with a flame-ionization detector and Ultra 1
capillary column (25 m × 0.32 mm i.d. × 0.52 µm film thick-
ness, 100% dimethyl polysiloxane; Hewlett-Packard). The
temperature program was chosen as follows: 170°C (5 min),
170 to 275°C (10°C/min), and 275°C (10 min). The injector
and detector temperatures were 250 and 280°C, respectively.
The carrier gas, N2, flow rate was 4.5 mL/min. The split was
25:1. For identification and quantification of unreacted fatty
acids, unreacted menthol and menthyl esters, at first mixtures
of these components in different concentrations were prepared
using standard fatty acids, standard menthol, and standard men-
thyl esters prepared according to the method of Wu et al. (7).
Later these mixtures were analyzed directly without derivatiz-
ing the fatty acids by capillary gas chromatography under the
same working conditions mentioned above in order to get re-
sponse factors of reactants and products and to establish cali-
bration curves for them. The response factors of fatty acids and
menthol esters were 1–0.96, respectively. However, because
the retention time of menthol was very close to that of solvent,
the response factor of menthol could not be measured. Since
the response factors of fatty acids and menthyl esters varied
over a rather small range, the extent of esterification was fol-
lowed by the amount of fatty acid consumed and the amount of
menthyl ester produced during the reaction, using just peak
areas and calibration curves. The percentage molar conversion
of fatty acids, which was not corrected for purity of fatty acid,
was defined as (mmol of reacted fatty acid/initial mmol of fatty
acid) × 100. Then the composition of the reaction mixtures was
calculated using the number of millimoles of reacted fatty
acids, the initial weights of reactants, and the mole weights of
reactants and menthol esters.
EXPERIMENTAL PROCEDURES
Materials. Commercial lipase from C. rugosa (Lipase AY
“Amano” 30) was a gift of Amano Pharmaceutical Co. Ltd.
(Nagoya, Japan) and was used as received. Its lipolytic activity
was determined as 39375 U/genzyme using olive oil as substrate
according to the method of Rosu and et al. (9). Lauric acid
(12:0, 91% pure), and 90.5% pure myristic acid (14:0) were pur-
chased from Hopkin & Williams Ltd. (Essex, England).
Palmitic acid (16:0, 97.5% pure) and 89.8% pure stearic acid
(18:0) were products of Fluka Chemie AG (Buchs, Switzer-
land). Margaric acid (17:0, 82% pure) and 77.5% pure oleic acid
(18:1) were obtained from Sigma (Deisenhofen, Germany) and
Merck Chemical Co. (Darmstadt, Germany), respectively.
(−)-Menthol (purity, 99.7%) was obtained from Haarmann &
Reimer GmbH (Holzminden, Germany). Isooctane (purity,
99.5%) was purchased from Carlo Erba reaganti (Milano, Italy).
Esterification reactions. Esterification reactions in isooctane
were carried out in a glass reaction flask (25 mL) that was
placed in a water bath in duplicate. The presented data are the
averages of duplicate determinations. Heating of the water bath
and stirring of the reaction mixture were performed with a mag-
netic stirrer equipped with heating unit (Framo-Geraetetechnik
M22/1 5655; Franz Morat KGaA, Eisenbach, Germany). The
stirring rate was adjusted to 500 rpm and the reaction tempera-
ture was kept constant with an accuracy of ±1°C by a tempera-
ture controller. In all reactions, the initial (−)-menthol molarity
was 200 mM. The molar ratios of (−)-menthol/fatty acid were
varied at 2:1, 1:1, and 1:2 in 10 mL isooctane. The reaction mix-
ture was heated to 35°C with stirring. The esterification reaction
was started by adding the enzyme to the reaction mixture, and
the 0 h sample (approximately 0.4 mL) was taken after 0.5 min.
For the removal of the generated water, 0.1 g of 4 Å molecular
sieves, previously dried at 120°C for 18 h, were added to the re-
action medium 1 h after the incubation started. Samples were
taken at selected time intervals and heated in a water bath at
RESULTS AND DISCUSSION
Effect of enzyme amount on the fatty acid conversion. To study
the effect of enzyme amount on the esterification of
(−)-menthol with lauric acid a set of reactions was conducted
at a molar ratio of menthol to lauric acid of 2:1 in 10 mL isooc-
tane using 0.053, 0.5, 1.067, and 1.5 g of Lipase AY “Amano”
30/g (−)-menthol at 35°C for 72 h. As one observes in Figure
1, at the enzyme ratio of 0.053 g/g, (−)-menthol esterification
practically did not occur. At the enzyme concentration of 0.5
g/g (−)-menthol, the fatty acid molar conversion reached ap-
proximately 11% by 10 h and remained steady out to at least
72 h. Kamiya et al. (4) recently observed similar behavior using
the same lipase. However, the fatty acid conversion increased
JAOCS, Vol. 78, no. 1 (2001)

ENZYMATIC ESTERIFICATION OF (−)-MENTHOL
55
FIG. 1. Effect of enzyme amount on the esterification of (−)-menthol
with lauric acid. Esterification reactions were conducted at 35°C with a
mixture of 200 mM (−)-menthol and 100 mM lauric acid in isooctane.
E, Lipase AY “Amano” 30; M, (−)-menthol.
FIG. 2. Effect of temperature on Lipase AY “Amano” 30-catalyzed ester-
ification of (−)-menthol with lauric acid at molar ratio of 2:1
(mmol/mmol) in isooctane. Enzyme concentration was 1.5 g enzyme/g
(−)-menthol. Initial menthol concentration: 200 mM.
sharply when the enzyme concentration was increased to the was narrow and that the optimal temperature was around 35°C.
range of 1–1.5 g enzyme/g (−)-menthol. By using 1.5 g en- To compare our results with those in the literature, 35°C was
zyme/g (−)-menthol, the lauric acid molar conversion reached chosen as the reaction temperature in further experiments.
approximately 97% in 24 h. Hence, 1.5 g enzyme/g (−)-men-
Effect of fatty acid type on the esterification of (−)-menthol.
thol was chosen as the enzyme amount for further experiments. The effect of the fatty acid type on the esterification of (−)-men-
Table 1 shows changes in the composition of the ester products thol catalyzed by Lipase AY “Amano” 30 was investigated
during the reaction conducted using 1.5 g enzyme/g (−)-men- using saturated fatty acids (lauric, myristic, palmitic, stearic,
thol as a function of the reaction time. Most of the changes oc- margaric and the unsaturated fatty acid oleic acid at a 2:1
curred between 0 and 24 h.
(−)-menthol/fatty acid molar ratio for 48 h. As can be seen in
Effect of the temperature on the esterification of (−)-men- Figure 3, except for the reaction using myristic acid, the con-
thol with lauric acid. The effect of temperature on the esterifi- versions exhausted the supply of fatty acid between 10 and 24 h
cation of menthol with lauric acid was studied with reactions of incubation. The lowest and the highest conversion rates were
conducted at a molar ratio of 2:1 (alcohol/acid) at temperatures obtained with myristic acid and margaric acid, respectively. The
ranging from 25 to 45°C. As shown in Figure 2, the conversion long-chain fatty acids with an even number of carbon atoms
of lauric acid was practically the same between 25 and 35°C. were converted at higher reaction rates than the even-numbered
Kamiya et al. (4) reported that the optimal reaction tempera- shorter-chain fatty acids, and the 14:0 fatty acid resulted in the
ture region for the coated-lipase originating from C. rugosa lowest percentage conversion. These results are in agreement
with the results of Kamiya et al. (4), who concluded that a more
hydrophobic substrate is preferred by surfactant-coated lipases.
TABLE 1
We also observed that margaric acid and oleic acid were con-
verted to their menthyl esters at higher molar conversions than
the saturated fatty acids with an even number of carbon atoms.
These results suggest that the fatty acid with an odd number of
carbon atoms and the unsaturated fatty acid may be preferred
substrates of Lipase AY “Amano” 30, resulting in higher sub-
strate selectivity than the even-numbered saturated fatty acids.
The initial rates of esterification of lauric, myristic, palmitic,
stearic, margaric, and oleic acids were found to be 14, 10, 25,
19, 38, and 30 µmol/min/g of enzyme, respectively. The reason
for the lower initial rate of the esterification reaction conducted
with stearic acid than that of palmitic acid may be dependent on
the lower stearic acid purity (89.8%), since stearic acid contains
8.8% saturated fatty acids with lower carbon numbers, such as
12:0, 14:0, and 16:0 acids. However, margaric and oleic acids
were less pure than stearic acid, yet were better substrates,
which may be related to their chemical natures.
Effect of Reaction Time on the Composition of Ester Productsa
Composition of ester product (wt%)
Reaction time (h)
(−)-Menthol
Fatty acids
Menthyl esters
0
1
1.5
2
3
4
5
6
7
8
60.8
58.7
57.7
56.6
55.0
52.6
51.2
49.1
47.6
45.9
32.5
32.2
32.2
39.0
36.2
34.9
33.3
31.3
28.0
26.2
23.4
21.3
19.1
1.3
0.2
5.1
7.4
10.1
13.7
19.4
22.6
27.5
31.1
35.0
66.2
67.0
67.0
24
48
72
0.8
0.8
aReaction was performed in 10 mL isooctane with a 2:1 mole ratio of (−)-men-
thol to lauric acid using 1.5 g Lipase AY “Amano” 30 per g of menthol, 0.1 g
molecular sieves, and a menthol concentration of 200 mM at 35°C.
JAOCS, Vol. 78, no. 1 (2001)

56
B. BABALI ET AL.
tively. The compositions of the ester products at 48 h at molar
ratios of 2:1 and 1:1 (−)-menthol to lauric acid were 32.2%
(−)-menthol, 0.8% fatty acid, 67% menthyl esters, and 3.2%
(−)-menthol, 4.1% fatty acid, and 92.7% menthyl esters, re-
spectively. According to the compositions of ester products, the
optimal molar ratio of (−)-menthol to lauric acid was estab-
lished as 1:1 for the resolution of racemic menthol.
The data presented here show that Lipase AY “Amano” 30
from C. rugosa without a surfactant coating could be used suc-
cessfully for the esterification of (−)-menthol with fatty acids
in solvent at both high menthol and enzyme concentrations. In
general, the molar conversion of fatty acids reached approxi-
mately 98% after 48 h. With margaric acid, the molar conver-
sion was found to be about 98% after 8 h. This fact may be re-
lated to a possible higher affinity of lipase AY “Amano” 30 for
fatty acids with an odd number of carbon atoms. To test this
hypothesis, further experiments should be carried out using
other odd-numbered fatty acids
FIG. 3. Effect of fatty acid type on the fatty acid molar conversion. Re-
actions were conducted at 35°C and 2:1 molar ratio of (−)-menthol/fatty
acid in isooctane using Lipase AY “Amano” 30 [1.5 g/g (−)-menthol].
Initial menthol concentration: 200 mM.
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FIG. 4. Effect of molar ratio on the esterification of (−)-menthol with
lauric acid. Reactions were conducted at 35°C in isooctane using Li-
pase AY “Amano” 30 [1.5 g/g (−)-menthol]. LA, lauric acid; see Figure
1 for other abbreviation. Initial menthol concentration fixed at 200 mM
in all reactions.
Effect of molar ratio on the esterification of (−)-menthol.
The esterification reactions were carried out with 1, 2, and 4
mmol lauric acid, keeping the (−)-menthol content at 2 mmol.
Figure 4 shows that the (−)-menthol/lauric acid molar ratio af-
fected the rate of reaction. The highest reaction rate and con-
version of fatty acid were obtained with the molar ratio of 2:1
(−)-menthol to lauric acid. After 48 h the conversions of lauric
acid at molar ratios of 2:1 and 1:1 reached 98 and 93%, respec-
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Moriyama, Y. Tominaga, and T. Terai, Enzymatic Synthesis of
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[Received April 13, 2000; accepted September 25, 2000]
JAOCS, Vol. 78, no. 1 (2001)