Enzymatic esterification of (-)-menthol with lauric acid in isooctane by sorbitan monostearate-coated lipase from Candida rugosa

Article abstract of DOI:10.1007/s11746-001-0239-6

Esterification of (-)-menthol and (±)-menthol with lauric acid in isooctane was successfully catalyzed by a commercial nonionic surfactant (sorbitan monostearate)-coated lipase from Candida rugosa (Lipase AY "Amano" 30) at the molar ratio of 1:1 andat 35°

Full text of DOI:10.1007/s11746-001-0239-6

−
Enzymatic Esterification of ( )-Menthol with Lauric Acid
in Isooctane by Sorbitan Monostearate-Coated Lipase
from Candida rugosa
Banu Babali, H. Ayse Aksoy*, Melek Tuter, and Guldem Ustun
Istanbul Technical University, Chemical Engineering Department, 80626, Maslak, Istanbul, Turkey
tem (7). Therefore we aimed to evaluate the usefulness of a
commercial nonionic surfactant “Sorbitan monostearate”-
coated lipase from C. rugosa (Lipase AY “Amano” 30) for
the esterification of (−)-menthol, and to compare its activity
toward this substrate with that of unmodified enzyme. Fur-
thermore, we aimed to show that optical resolution of racemic
menthol can be carried out by esterification catalyzed by this
preparation.
ABSTRACT: Esterification of (−)-menthol and ( )-menthol with
lauric acid in isooctane was successfully catalyzed by a com-
mercial nonionic surfactant (sorbitan monostearate)-coated li-
pase from Candida rugosa (Lipase AY “Amano” 30) at the molar
ratio of 1:1 and at 35°C using 1.5 g enzyme/g (−)-menthol and
0.1-g molecular sieves. After 1 h, molar conversion of (−)-men-
thol reached 81%. Equilibrium was reached after ca. 4 h, giving
a (−)-menthol molar conversion of 94%. Under the same condi-
tions, native lipase catalyzed the esterification of (−)-menthol
and lauric acid to yield a molar conversion of 93% after 72 h.
Coating the lipase with sorbitan monostearate increased the es-
terification rates of both (−)-menthol and ( )-menthol with lau-
ric acid. After 6 h, the molar conversions of (−)-menthol and ( )-
menthol were 94, and 62%, respectively.
EXPERIMENTAL PROCEDURES
Materials. Commercial lipase from C. rugosa (formerly C.
cylindracea), Lipase AY “ Amano” 30, was a gift of Amano
Pharmaceutical Co. Ltd. (Nagoya, Japan) and was used as re-
ceived. Its lipolytic activity was determined as 39,375 U/g en-
zyme using olive oil as substrate according to the method of
Rosu et al. (8). Lauric acid (12:0) of 91% purity was pur-
chased from Hopkin & Williams Ltd. (Essex, England). (−)-
Menthol (purity, 99.7%) and ( )-menthol (purity, 99%) were
obtained from Haarmann & Reimer GmbH (Holzminden,
Germany). Isooctane (purity, 99.5%) was purchased from
Carlo Erba reaganti (Milano, Italy). Dehymuls SMS (sorbitan
monostearate) was a gift of Henkel Co. (Istanbul, Turkey).
Preparation of surfactant-coated enzyme. Surfactant-
coated lipase was prepared using the method of Basheer et al.
(7). Lipase AY (3 g) was dissolved in 1 L Tris buffer (pH 5.5)
and magnetically stirred at 10°C. Sorbitan monostearate in
ethanol (0.75 g/20 mL) was added dropwise to the stirred en-
zyme solution and sonicated in an ultrasonic bath for 10 min.
After magnetically stirring for 3 h at 10°C, surfactant-coated
enzyme was precipitated by adding acetone at 10°C, collected
by centrifugation (21,000 × g) and then air-dried. A yellow-
ish powder (the surfactant-coated lipase) was obtained with a
yield of ca. 13% mass and lipolytic activity of 67,828.7 U/g
surfactant-coated lipase.
Paper no. J9710 in JAOCS 78, 173–175 (February 2001).
KEY WORDS: Candida rugosa lipase, esterification, lauric acid,
menthol, sorbitan monostearate.
Menthol (p-menthan-3-ol) is a secondary terpene alcohol and
has eight optically active isomers with different organoleptic
properties. (−)-Menthol has a characteristic peppermint fla-
vor and refreshing coolness. Because of its flavor and refresh-
ing coolness, (−)-menthol is widely used in foods, cosmetics,
and pharmaceuticals (1). Enzymatic esterification of (−)-
menthol is a highly selective method for the resolution of
racemic menthol, which is an equal molar mixture (+)- and
(−)-menthol. Lipases of different origins have been examined
in water–oil emulsions (2,3) and in organic solvents (4–6) to
elucidate the effects of temperature, solvents, and fatty acid
chain lengths. Kamiya et al. (4) investigated the enantiose-
lective esterification of (−)-menthol with lauric acid in isooc-
tane at 35°C using six different lipases. Among them, only
the lipase from Candida cylindracea (Lipase AY) catalyzed
the esterification of (−)-menthol with lauric acid. Moreover,
coating Lipase AY with a nonionic surfactant (glutamic acid
dioleyl ester ribitol amide) increased the catalytic activity of
the lipase. The rate of esterification of (−)-menthol with lau-
ric acid catalyzed by surfactant-coated Lipase AY was more
than 100 times that of the native lipase.
Esterification reactions. Esterification reactions in isooc-
tane were carried out in a glass reaction flask (25 mL) at the
optimal conditions determined for native Lipase AY in our
previous study in duplicate (9). Heating of the water bath and
stirring of the reaction mixture were performed with a mag-
netic stirrer equipped with a heating unit (Framo-Geraetetech-
nik M22/1 5655; Franz Morat KG, Eisenbach, Germany). The
stirring rate was adjusted to 500 rpm, and the reaction tem-
Sorbitan monostearate-coated Rhizopus japonicus lipase
has been shown to catalyze the esterification of long-chain
fatty acids and fatty alcohols in a microaqueous n-hexane sys-
*To whom correspondence should be addressed. E-mail: aksoyha@itu.edu.tr perature was 35°C and kept constant with an accuracy of 1°C
Copyright © 2001 by AOCS Press 173 JAOCS, Vol. 78, no. 2 (2001)

174
B. BABALI ET AL.
by a temperature controller. The esterification reactions were
initiated by adding enzyme (59,000 U/g menthol) either in
original or surfactant-coated form to 10 mL isooctane that
contained 200 mM menthol and 200 mM lauric acid at the re-
action temperature. The 0-h sample (ca. 0.4 mL) was taken
after 0.5 min. For the removal of the generated water, 0.1 g of
4 Å molecular sieves, which were dried at 120°C for 18 h,
were added to the reaction medium 1 h after the incubation
started. Samples were taken at selected time intervals and
heated in a water bath at 90°C for 15 min to inactivate the en-
zyme and then centrifuged to separate the ester product. The
ester products were dried with anhydrous Na2SO4 and ana-
lyzed by capillary gas chromatography.
Analytical methods. The composition of the esterification
product was determined in a capillary gas chromatography
apparatus, HP 5890 Series II (Hewlett-Packard, Waldron,
Germany) equipped with a flame-ionization detector and
Ultra 1 capillary column (25 m × 0.32 mm i.d. × 0.52 µm film
FIG. 1. Time course of the esterification of (−)-menthol and ( )-menthol
with lauric acid by the lipase from Candida rugosa. Reaction conditions:
200 mM (−)-menthol or 200 mM ( )-menthol and 200 mM lauric acid
in 10 mL isooctane, 35°C, 59,000 U/g (−)-menthol.
thickness, 100% dimethyl polysiloxane; Hewlett-Packard). conditions mentioned above. Figures 1 and 2 show the time
The temperature program was chosen as follows: 170°C (5 courses of the esterification reactions of both (−)-and ( )-
min), 170 to 275°C (10°C/min), and 275°C (10 min). The in- menthol with lauric acid using the lipase from C. rugosa, Li-
jector and detector temperatures were 250 and 280°C, respec- pase AY “Amano” 30, and sorbitan monostearate-coated li-
tively. The carrier gas, N2, flow rate was 4.5 mL/min. The pase, respectively. The molar conversion of ( )-menthol to its
split was 25:1.
corresponding ester during a 6-h incubation was ca. 19%
when the lipase in original form was used, compared to more
than 62% when the lipase modified with sorbitan mono-
stearate was used (Figs. 1 and 2). Under the same reaction
RESULTS AND DISCUSSION
Effect of the modified enzyme on the esterification reaction of conditions, after 6 h, molar conversion of 94% was obtained
(−) menthol. Figures 1 and 2 show the time courses of the (−)- with (−)-menthol when the reaction was conducted using sor-
menthol conversion catalyzed by lipase in original and sorbi- bitan monostearate-coated lipase while the corresponding
tan monostearate-coated forms at 35°C. As previously men- molar conversion was only 23% using the lipase in original
tioned by Kamiya et al. (4), surfactant-coated lipase was sol- form (Figs. 1 and 2). Moreover, the lipase in original form
uble in anhydrous organic solvent and showed high catalytic catalyzed the esterification of (−)-menthol with an equilib-
activity. Indeed it was observed that after 1-h reaction, molar rium molar conversion of 93.1% and that of ( )-menthol with
conversion of (−)-menthol reached 81% in the esterification an equilibrium molar conversion of 58.3%, following 72-h
reaction catalyzed with sorbitan monostearate-coated lipase incubation (Fig. 1).
(Fig. 2). The equilibrium conversion was reached after ca. 4
h, giving a (−)-menthol molar conversion of 94%. However,
the conversion of (−)-menthol was only 77% following 24-h
incubation when the lipase in original form was used (Fig. 1).
It was recently reported by Kamiya et al. (4) that in the ester-
ification reaction of 5 mM (−)-menthol and 10 mM lauric acid
in 10 mL isooctane at 35°C using glutamic acid dioleyl ester
ribitol amide-coated lipase from C. cylindracea, more than
90% of (−)-menthyl laurate was produced within 24 h. In
comparing our results with those obtained by Kamiya et al.
(4), it is very important to point out that the reaction between
(−)-menthol and lauric acid could readily reach more than
90% after 4 h instead of 24 h by coating the same lipase with
sorbitan monostearate. This can be explained to some degree
by the different natures of the surfactants used.
Effect of the modified enzyme on the esterification reaction
of ( )-menthol. In order to investigate the effect of coating C.
rugosa lipase with sorbitan monostearate on the esterification
of (−)- and ( )-menthol with lauric acid, these reactions were
conducted separately in isooctane under the same reaction
FIG. 2. Time course of the esterification of (−)-menthol and ( )-menthol
with lauric acid by sorbitan monostearate coated-lipase from C. rugosa.
Reaction conditions: 200 mM (−)-menthol or 200 mM ( )-menthol and
200 mM lauric acid in 10 mL isooctane, 35°C, 59,000 U/g (−)-menthol.
See Figure 1 for abbreviations.
JAOCS, Vol. 78, no. 2 (2001)

ENZYMATIC ESTERIFICATION OF (−)-MENTHOL BY SURFACTANT-COATED LIPASE
175
4. Kamiya, N., M. Goto, and F. Nakashio, Surfactant-Coated Li-
pase Suitable for the Enzymatic Resolution of Menthol as a Bio-
catalyst in Organic Media, Biotechnol. Prog. 11:270–275 (1995).
5. Kamiya, N., and M. Goto, How Is Enzymatic Selectivity of Men-
thol Esterification Catalyzed by Surfactant-Coated Lipase Deter-
mined in Organic Media?, Ibid. 13:488–492 (1997).
6. Wu, W.H., C.C Akoh, and R.S. Phillips, Lipase-Catalyzed Stere-
oselective Esterification of DL-Menthol in Organic Solvents
Using Acid Anhydrides as Acylating Agents, Enzyme Microb.
Technol. 18:536–539 (1996).
Thus, the data presented here show that sorbitan mono-
stearate-coated lipase from C. rugosa (Lipase AY “Amano”
30) is a highly effective biocatalyst which can be used for the
esterification of (−)-menthol and possibly for the resolution
of ( )-menthol in a considerably shorter reaction time (4 h)
than feasible when using the same enzyme not treated with
sorbitan monstearate.
7. Basheer, S., U. Cogan, and M. Nakajima, Esterification Kinetics
of Long-Chain Fatty Acids and Fatty Alcohols with a Surfactant-
Coated Lipase in Hexane, J. Am. Oil Chem. Soc. 76:1785–1789
(1998).
8. Rosu, R., Y. Uozaki, Y. Iwasaki, and T. Yamane, Repeated Use
of Immobilized Lipase for Monoacylglycerol Production by
Solid-Phase Glycerolysis of Olive Oil, Ibid. 74:445–450 (1997).
9. Babali, B., H.A. Aksoy, M. Tüter, and G. Ustun, Enzymatic Es-
terification of (−)-Menthol with Fatty Acids in Solvent by a Com-
mercial Lipase from Candida rugosa, Ibid. 78:53–56 (2001).
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[Received July 24, 2000; accepted November 14, 2000]
JAOCS, Vol. 78, no. 2 (2001)