Steryl and stanyl esters of fatty acids by solvent-free esterification and transesterification in vacuo using lipases from Rhizomucor miehei, Candida antarctica, and Carica papaya

Article abstract of DOI:10.1021/jf0107407

Sitostanol has been converted in high to near-quantitative extent to the corresponding long-chain acyl esters via esterification with oleic acid or transesterification with methyl oleate or trioleoylglycerol using immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (lipase B, Novozym 435) as biocatalysts in vacuo (20-40 mbar) at 80 °C, whereas the conversion was markedly lower at 60 and 40 °C. Corresponding conversions observed with papaya (Carica papaya) latex lipase were generally lower. High conversion rates observed in transesterification of sitostanol with methyl oleate at 80 °C using Lipozyme IM were retained even after 10 repeated uses of the biocatalyst. Saturated sterols such as sitostanol and 5α-cholestan-3β-ol were the preferred substrates as compared to Δ⁵-unsaturated cholesterol in transesterification reactions with methyl oleate using Lipozyme IM. Transesterification of cholesterol with diethyl 1,8-octanedioate using Lipozyme IM in vacuo yielded methylcholesteryl 1,8-octanedioate (75%) and dicholesteryl 1,8-octanedioate (5%). However, transesterification of cholesterol with diethyl carbonate and that of oleyl alcohol with ethylcholesteryl carbonate, both catalyzed by Lipozyme IM, gave ethylcholesteryl carbonate and oleylcholesteryl carbonate, respectively, in low yield (20%). Moreover, cholesterol was transesterified with ethyl dihydrocinnamate using Lipozyme IM to give cholesteryl dihydrocinnamate in moderate yield (56%), whereas the corresponding reaction of lanosterol gave lanosteryl oleate in low yield (14%).

Full text of DOI:10.1021/jf0107407

5210
J. Agric. Food Chem. 2001, 49, 5210−5216
Ster yl a n d Sta n yl Ester s of F a tty Acid s by Solven t-F r ee
Ester ifica tion a n d Tr a n sester ifica tion in Va cu o Usin g Lip a ses fr om
Rh izom u cor m ieh ei, Ca n d id a a n ta r ctica , a n d Ca r ica pa pa ya
Nikolaus Weber,* Petra Weitkamp, and Kumar D. Mukherjee
Institute for Biochemistry and Technology of Lipids, H. P. Kaufmann-Institute, Federal Centre for Cereal,
Potato and Lipid Research, Piusallee 68, D-48147 Mu¨nster, Germany
Sitostanol has been converted in high to near-quantitative extent to the corresponding long-chain
acyl esters via esterification with oleic acid or transesterification with methyl oleate or trioleoyl-
glycerol using immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica
(lipase B, Novozym 435) as biocatalysts in vacuo (20-40 mbar) at 80 °C, whereas the conversion
was markedly lower at 60 and 40 °C. Corresponding conversions observed with papaya (Carica
papaya) latex lipase were generally lower. High conversion rates observed in transesterification of
sitostanol with methyl oleate at 80 °C using Lipozyme IM were retained even after 10 repeated
uses of the biocatalyst. Saturated sterols such as sitostanol and 5R-cholestan-3â-ol were the preferred
substrates as compared to ∆5-unsaturated cholesterol in transesterification reactions with methyl
oleate using Lipozyme IM. Transesterification of cholesterol with dimethyl 1,8-octanedioate using
Lipozyme IM in vacuo yielded methylcholesteryl 1,8-octanedioate (75%) and dicholesteryl 1,8-
octanedioate (5%). However, transesterification of cholesterol with diethyl carbonate and that of
oleyl alcohol with ethylcholesteryl carbonate, both catalyzed by Lipozyme IM, gave ethylcholesteryl
carbonate and oleylcholesteryl carbonate, respectively, in low yield (20%). Moreover, cholesterol
was transesterified with ethyl dihydrocinnamate using Lipozyme IM to give cholesteryl dihydro-
cinnamate in moderate yield (56%), whereas the corresponding reaction of lanosterol gave lanosteryl
oleate in low yield (14%).
Keyw or d s: Candida antarctica lipase B; Carica papaya lipase; esterification and transesterification
in vacuo; fatty acids; Rhizomucor miehei lipase; solvent-free esterification and transesterification;
stanyl esters; steryl esters
INTRODUCTION
the exent of formation of steryl esters is rather moderate
and the use of organic solvents limits the application of
such products.
Cholesteryl esters are widely used for technical ap-
plications such as liquid crystal display devices. Fatty
acid esters of sterols and steroids are well-known
ingredients of cosmetics, nutraceuticals, and pharma-
ceutical formulations (1). Plant steryl and stanyl esters
have been recently found to be effective in lowering
plasma cholesterol concentration by inhibiting the ab-
sorption of cholesterol from the small intestine (2-4).
Special margarines fortified with steryl esters are now
commercially available as functional foods with the
ability to reduce both total and low-density lipoprotein
(LDL) cholesterol levels (5).
Fatty acid esters of sterols, stanols, and steroids are
usually prepared from the corresponding sterols by
chemical esterification with fatty acids or interesteri-
fication with fatty acid methyl esters as well as by their
reaction with fatty acid halogenides or anhydrides (6).
Enzymatic procedures for the preparation of steryl
esters using organic solvents and molecular sieves or
other drying agents have been reported (1, 7-15).
Moreover, a method has been described for the prepara-
tion of steryl esters of polyunsaturated fatty acids in
an aqueous system (16). In most of the above methods
Recently, we described the enzymatic preparation of
carboxylic acid esters, particularly fatty acid esters, of
sterols, stanols, and steroids in high yield by esterifi-
cation and transesterification of fatty acids and other
carboxylic acid esters, respectively, with the 3-hydroxy
group of sterols, stanols, or steroids in vacuo at moder-
ate temperature using immobilized lipase from Candida
rugosa as the catalyst (17). Neither an organic solvent
nor a molecular sieve was required. Here we report the
synthesis of steryl and stanyl esters of carboxylic acids,
particularly fatty acids, in vacuo using immobilized
lipases from Rhizomucor miehei (Lipozyme IM) and
Candida antarctica (lipase B, Novozym 435) as well as
papaya (Carica papaya) latex under various conditions.
EXPERIMENTAL PROCEDURES
Ch em ica ls. Sitostanol (stigmastanol), methyl myristate,
methyl stearate, oleic acid, methyl oleate, triolein, ethyl
dihydrocinnamate, 1,8-octanedicarboxylic acid dimethyl ester
(suberic acid dimethylester), diethyl carbonate, cholesteryl
chloroformate, oleylcholesteryl carbonate, and oleyl alcohol
were obtained from Sigma-Aldrich-Fluka (Deisenhofen, Ger-
many). Cholesterol, 5R-cholestan-3â-ol (dihydrocholesterol),
lanosterol (containing ∼30% dihydrolanosterol), and 1-meth-
ylimidazole were purchased from Merck (Darmstadt, Ger-
many). N-Methyl-N-(trimethylsilyl)heptafluorobutyramide
* Author to whom correspondence should be addressed
(telephone +49-251-481 670; fax +49-251-519 275; e-mail
ibtfett@uni-muenster.de).
10.1021/jf0107407 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/27/2001

Lipase-Catalyzed Steryl Ester Preparation
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5211
F igu r e 1. Chemical structures of steryl and stanyl esters.
(MSHFBA) was a product of Macherey-Nagel (Du¨ren, Ger-
many). The immobilized lipase preparations from R. miehei
(Lipozyme IM 20; 23 batch interesterification units/g; 10% w/w
water) and Cd. antarctica (lipase B, Novozym 435; 10500
propyl laurate units/g; 2% w/w water) were kindly provided
by Novozymes (Bagsvaerd, Denmark). The granular papaya
(Cr. papaya) latex preparation (Sigma-Aldrich-Fluka) was
ground in a mortar with pestle to a fine powder to pass a 0.8
mm mesh width sieve. Ethylcholesteryl carbonate standard
was prepared by reacting 0.5 g (1.1 mmol) of cholesteryl
chloroformate dissolved in 3 mL of dichloromethane with 7
mL (150 mmol) of water-free methanol in the presence of 0.2
g of 4-(dimethylamino)pyridine (Sigma-Aldrich-Fluka) for 4 h
at 45 °C. The reaction product was purified by column
chromatography as described below.
Lip a se-Ca ta lyzed Rea ction s. Unless stated otherwise
fatty acid or fatty acid methyl ester, 300 µmol each, or
trioleoylglycerol (200 µmol) was esterified or transesterified
with sitostanol or cholesterol, 100 µmol each, in the presence
of 50 mg of each lipase preparation by magnetic stirring in a
screw-capped tube in vacuo at 40, 60, and 80 °C for various
periods with water (or methanol)-trapping in the gas-phase
using KOH pellets. The vacuum used was 20-40 mbar,
measured at room temperature.
lipase at a similar ratio of biocatalyst to substrate mixture as
in the foregoing reaction.
Transesterification reactions of cholesterol (100 µmol) with
ethyl dihydrocinnamate (initially 1 mmol, followed by ad-
ditional 0.5 mmol after 32 h) and lanosterol (100 µmol) with
methyl oleate (600 µmol) were performed in the presence of
100 mg of Lipozyme IM for 96 h in vacuo at 80 °C.
Transesterification of cholesterol (100 µmol) with dimethyl
1,8-octanedioate (400 µmol, two 200 µmol portions added
initially and after 8 h) was carried out in the presence of 100
mg of Lipozyme IM for 72 h in vacuo at 80 °C. Transesterifi-
cation of cholesterol (100 µmol) with diethyl carbonate (4.1
mmol) was carried out at 400 mbar and 60 °C in the presence
of 50 mg of molecular sieve 4 Å and 50 mg of Lipozyme IM for
48 h. Transesterification of ethylcholesteryl carbonate with
oleyl alcohol (300 µmol) was performed using 50 mg of
Lipozyme IM at 20 mbar and 80 °C for 48 h.
Th in -La yer Ch r om a togr a p h y (TLC). Aliquots of the
reaction mixtures were checked for conversion by TLC on 0.3
mm layers of Silica Gel H (Merck, Darmstadt, Germany) using
isohexane/diethyl ether (95:5, v/v); spots were located by iodine
staining. Alternatively, the TLC plates were sprayed with 30%
aqueous sulfuric acid and heated in an oven kept at 120 °C;
∆5-sterols and their esters appeared as red spots (Liebermann-
Burchard test). The Rf values of the various compounds were
as follows: fatty acid steryl and stanyl esters, 0.6-0.7; methyl
oleate, 0.4-0.5; trioleoylglycerol, 0.3-0.35; sterols and stanols,
0.1-0.15; oleic acid, <0.1.
In one set of experiments the reusability of the lipase
preparations was evaluated in the transesterification of sito-
stanol with methyl oleate as described above. In each case the
lipase was separated from the reaction mixture by repeated
extraction with diethyl ether and centrifugation. Subsequently,
the solvent was removed from the lipase in a stream of
nitrogen followed by evacuation at 40 °C for 2 h and weighing.
A fresh batch of reaction mixture was then added to the used
Reaction mixtures containing steryl esters of 1,8-octane-
dicarboxylic acid and carbonic acid were analyzed by TLC
on 0.3 mm layers of Silica Gel H using isohexane/diethyl
ether (4:1, v/v). The Rf values of the various compounds

5212 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Weber et al.
(Figure 1) were as follows: dicholesteryl 1,8-octanoate,
0.70-0.80; methylcholesteryl 1,8-octanedioate, 0.45-0.55; di-
methyl 1,8-octanoate, 0.30-0.40; 1,8-octanedioic acid, <0.1;
ethylcholesteryl carbonate, 0.60-0.65; oleylcholesteryl carbon-
ate, 0.75-0.80.
alcohol contained the heat sensitive oleylcholesteryl carbonate
(Figure 1); they were analyzed by RP-HPLC as follows. The
HPLC system consisted of a Merck-Hitachi pump L-6200 (E.
Merck) equipped with a Kontron (Kontron Instruments, Milan,
Italy) UV-vis HPLC 332 detector (set to a wavelength of 210
nm) and an ACS (Applied Chromatography Systems, Maccles-
field, U.K.) mass detector model 750/14 (thermostated to 55
°C), which were used in series. UV and mass traces were
monitored and evaluated in a KromaSystem 2000 data acqui-
sition unit (Kontron Instruments).
Ga s Ch r om a togr a p h y (GC). In esterification reactions
aliquots of reaction mixtures, ∼15 mg, were extracted twice
with 2 mL of diethyl ether, each. The ether extract was
concentrated and treated with a solution of diazomethane in
diethyl ether to convert the unreacted fatty acids to methyl
esters. The resulting mixture of methyl esters, unreacted
sterols or stanols, and fatty acid steryl or stanyl esters was
analyzed by GC. In transesterification reactions aliquots of
products consisting of fatty acid methyl esters or triacylglyc-
erols and unreacted sterols or stanols as well as carboxylic
acid steryl or stanyl esters were analyzed without derivatiza-
tion by GC. All GC samples, dissolved in dichloromethane,
were filtered through a 0.45 µm syringe filter before injection
into the gas chromatograph. A Hewlett-Packard (Bo¨blingen,
Germany) HP-5890 series II gas chromatograph equipped with
a flame ionization detector was used. Separations were carried
out on a 0.1 µm Quadrex 400-1HT (Quadrex Corp., New
Haven, CT) fused silica capillary column, 15 m × 0.25 mm
i.d., using hydrogen as the carrier gas (column pressure ) 50
kPa) initially at 160 °C for 2 min, followed by linear program-
ming from 160 to 180 °C at 5 °C‚min-1 and from 180 to 410
°C at 20 °C‚min-1; the final temperature of 410 °C was held
for 10 min. The split ratio was 1:10; the injector as well as
flame ionization detector temperature was 350 °C. Peaks in
gas chromatograms were assigned by comparison of their
retention times with those of commercially available standards
or those prepared by chemical or enzymatic synthesis. Peak
areas and percentages were calculated using a Hewlett-
Packard 3365 series GC ChemStation software and corrected
using standard curves. The retention times of the various
compounds were as follows: methyl myristate, 0.9 min; methyl
stearate, 1.9 min; methyl oleate, 1.8 min; ethyl dihydrocin-
namate, 1.0 min; cholesterol and 5R-cholestan-3â-ol, 8.9 min;
sitostanol, 9.6 min; lanosterol, 9.5 min; dihydrolanosterol, 9.3
min; cholesteryl myristate, 12.9 min; cholesteryl stearate, 13.8
min; cholesteryl oleate, 13.9 min; cholesteryl dihydrocin-
namate, 13.9 min; sitostanyl oleate, 14.3 min; lanosteryl oleate,
14.2 min; dihydrolanosteryl oleate, 14.1 min; ethylcholesteryl
carbonate, 10.0 min; 1,8-octanedicarboxylic acid dimethyl ester
(suberic acid dimethyl ester), 0.9 min; 1,8-octanedicarboxylic
acid methyl cholesteryl diester (suberic acid methyl cholesteryl
diester), 12.0 min. Percentage conversions were determined,
unless stated otherwise, as the amount of product formed from
the corrected peak areas of the chromatograms of the mixtures
of steryl/stanyl esters and unreacted sterol/stanol.
Cholesterol and 5R-cholestan-3â-ol are not separated under
the GC conditions described above. Therefore, products
(∼5-10 mg) of Lipozyme IM-catalyzed transesterifications of
methyl oleate with a mixture of cholesterol and 5R-cholestan-
3â-ol, reacted under competitive conditions (i.e., both sterols
present in the reaction mixture), were silylated for GC using
100 µL of MSHFBA reagent in the presence of 5 µL of 1-meth-
ylimidazole at 110 °C for 10 min. After cooling, the reagents
were removed in a stream of nitrogen and the residual mixture
was dissolved in dichloromethane for GC injection. Separations
were carried out on a 0.25 µm SE-54 CB (CS-Chromatographie-
Service, Langerwehe, Germany) fused silica capillary column,
25 m × 0.25 mm i.d., coupled with a 0.52 µm HP-1 (Hewlett-
Packard) fused silica capillary column, 25 m × 0.32 mm i.d.,
using nitrogen as the carrier gas (column pressure ) 80 kPa).
Temperature was initially kept at 240 °C for 2 min, followed
by linear programming from 240 to 300 °C at 5 °C‚min-1; the
final temperature of 300 °C was held for 25 min. The retention
times of the two silylated sterols were as follows: cholesterol
derivative, 33.6 min; 5R-cholestan-3â-ol derivative, 34.2 min.
The amounts of unreacted cholesterol and 5R-cholestanol-3â-
ol were determined by GC to calculate the conversion.
Rever sed -P h a se High -P er for m a n ce Liqu id Ch r om a -
togr a p h y (RP -HP LC). The products of lipase-catalyzed
transesterification of ethylcholesteryl carbonate with oleyl
Diethyl carbonate was removed from aliquots of the reaction
mixtures by evaporation in a stream of nitrogen at 45 °C. The
remaining products were resuspended in 1 mL of acetone, and
lipase catalyst was separated by centrifugation followed by
filtration through a syringe filter (pore size ) 0.35 µm). The
resulting solution containing ethylcholesteryl carbonate, oleyl
alcohol, and oleylcholesteryl carbonate was concentrated and
analyzed using a 250 × 4 mm Hibar RP-18 colum, packed with
10 µm LiChrospher 100 (Merck), using acetonitrile/acetone
(25:75, v/v) as eluent at a flow rate of 1 mL/min. Injections
(∼1 mg of reaction mixture) were carried out with a Rheodyne
7161 sample injector (Cotati, CA) equipped with a 20 µL
sample loop. Synthetic and commercial standards were used
for comparison. The retention times of the various compounds
were as follows: oleyl alcohol, 3.8 min; ethylcholesteryl
carbonate, 7.3 min; oleylcholesteryl carbonate, 22.1 min.
Similarly, the products of lipase-catalyzed transesterifica-
tion of cholesterol with 1,8-octanedicarboxylic acid dimethyl
ester were analyzed by RP-HPLC (due to difficulties in the
determination of high molecular weight 1,8-octanedicarboxylic
acid dicholesteryl ester by high-temperature GC) using pro-
grammed elution starting with acetonitrile/acetone (25:75, v/v)
for 15 min, followed by linear increase of the acetone concen-
tration to a ratio of acetonitrile/acetone of 1:9 (v/v) within 5
min, followed by an isocratic period of 30 min. The retention
times of the various compounds (Figure 1) were as follows:
1,8-octanedicarboxylic acid dimethyl ester, 2.1 min; cholesterol,
13.1 min; 1,8-octanedicarboxylic acid methylcholesteryl di-
ester, 7.1 min; 1,8-octanedicarboxylic acid dicholesteryl ester,
36.1 min.
P u r ifica tion of Ster yl a n d Sta n yl Ester s by Colu m n
Ch r om a togr a p h y. The reaction mixtures resulting from
lipase-catalyzed esterification and transesterification of oleic
acid and methyl oleate, respectively, with sitostanol were
purified by column chromatography on silica gel using a 20 ×
1.5 cm glass column and mixtures of isohexane/diethyl ether
as eluents as described recently (17).
Similarly, the reaction mixtures resulting from Lipozyme
IM-catalyzed esterification of 1,8-octanedicarboxylic acid di-
methyl ester with cholesterol were purified by column chro-
matography as described above; 1,8-octanedicarboxylic acid
dicholesteryl ester and 1,8-octanedicarboxylic acid methylcho-
lesteryl diester eluted successively with 20 mL of isohexane/
diethyl ether (4:1, v/v) followed by 20 mL of isohexane/diethyl
ether (1:1, v/v). The two reaction products, that is, 1,8-oc-
tanedicarboxylic acid dicholesteryl ester and 1,8-octanedicar-
boxylic acid methylcholesteryl diester, present in the com-
bined fractions were separated by silica gel column chroma-
tography using three 10 mL fractions of isohexane/diethyl
ether (4:1, v/v).
Oleyl cholesteryl carbonate and methyl cholesteryl carbon-
ate were separated from the reaction mixture by column
chromatography as described above using isohexane/diethyl
ether (9:1, v/v) followed by isohexane/diethyl ether (4:1, v/v)
as eluents.
Meltin g P oin ts. Melting points (mp) of steryl esters
determined with a Thermovar heating block (Reichert, Vienna,
Austria) were as follows: sitostanyl oleate, 44-45 °C; 5R-
cholestan-3â-yl oleate, 30-31 °C; cholesteryl myristate, 85 °C
(86 °C) (18); cholesteryl stearate, 83-84 °C (82.5 °C) (18);
cholesteryl oleate, 48-50 °C (51 °C) (19); cholesteryl dihydro-
cinnamate, 111-113 °C (Sigma-Aldrich Fine Chemicals catalog
2000/2001, Taufkirchen, Germany, 109.5-110.5 °C); 1,8-
octanedicarboxylic acid methylcholesteryl diester, 83-84 °C;
1,8-octanedicarboxylic acid dicholesteryl ester, 188-191 °C

Lipase-Catalyzed Steryl Ester Preparation
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5213
(179.5 °C) (18); oleyl cholesteryl carbonate 32-33 °C (mp of
authentic sample ) 34-35 °C).
RESULTS AND DISCUSSION
Esterification and interesterification reactions, cata-
lyzed by lipases, have been widely used for bioorganic
synthesis and biotransformation of fats and other lipids;
however, little is known so far on the application of such
reactions in the preparation of short- and long-chain
cyclic or polyfunctional acyl esters of sterols, stanols,
and steroids (8, 20). We show here that esterification
and, particularly, transesterification reactions, cata-
lyzed by lipases from R. miehei (Lipozyme IM), Cd.
antarctica (lipase B, Novozym 435), and papaya (Cr.
papaya) latex in vacuo, provide fatty acyl esters of
sterols and stanols in high to near-quantitative conver-
sions. Neither an organic solvent nor water is added to
the reaction mixtures, and no drying agent such as
molecular sieve is used (with the exception of cholesteryl
carbonate formation).
Figure 2 shows the time course of formation of
sitostanyl oleate via esterification of oleic acid (Figure
2A) or transesterification of methyl oleate (Figure 2B)
with sitostanol (molar ratio stanol/acyl donor ) 1:3)
catalyzed by various amounts (6.25, 12.5, 25 and 50 mg)
of different lipases, that is, papaya lipase, Novozym 435,
and Lipozyme IM, in vacuo at 40, 60, and 80 °C. It is
evident from the results shown in Figure 2A that
highest conversion by esterification (∼89%, cf. Table 1)
is achieved at 80 °C in vacuo using Novozym 435 as
catalyst, whereas conversions at 40 and 60 °C are
markedly decreased. Slightly reduced conversions by
esterification are observed in the presence of Lipozyme
IM, whereas the conversions in the presence of papaya
lipase are generally lower. It is also obvious from Figure
2A that the extent of conversion increases with increas-
ing proportion of lipases and increasing temperature.
The results given in Figure 2B demonstrate that
higher conversions are achieved by transesterification
in vacuo using Lipozyme IM, Novozym 435, and papaya
lipase than by the corresponding esterification reactions
(Figure 2A). Maximum conversions (∼93-96% after 24
h) are observed using methyl oleate or triolein as acyl
donors in the presence of Lipozyme IM (cf. Table 1). The
data given in Figure 2B show that also in transesteri-
fication the extent of reaction strongly increases with
increasing proportions of lipases and increasing tem-
perature.
Figure 3 demonstrates the effects of various acyl
donors and temperatures on the formation of sitostanyl
oleate by esterification of sitostanol with oleic acid and
by transesterification of sitostanol with methyl oleate
or triolein, catalyzed by Lipozyme IM in vacuo at 40,
60, and 80 °C for various periods. Obviously, under these
reaction conditions transesterification with methyl ole-
ate gives the highest conversion rates at all three tem-
peratures studied, followed by transesterification with
triolein, whereas esterification reaction with oleic acid
yields distinctly lower proportions of sitostanyl oleate.
The time course of the formation of sitostanyl oleate
by transesterification of sitostanol with methyl oleate
at various molar ratios, catalyzed by Lipozyme IM in
vacuo at 80 °C, shows that increasing the molar ratio
of methyl oleate from 1:1 to 1:3 and finally 1:5 does not
markedly increase the extent of transesterification
(Figure 4). Under similar conditions, esterification of
sitostanol with oleic acid catalyzed by Candida rugosa
F igu r e 2. Time course of the formation of sitostanyl oleate
via (A) esterification of 300 µmol of oleic acid or (B) transes-
terification of 300 µmol of methyl oleate with 100 µmol, each,
of sitostanol catalyzed by various amounts (], 6.25 mg; O, 12.5
mg; 0, 25 mg; ×, 50 mg) of different lipases [left, papaya (Cr.
papaya) latex; center, Cd. antarctica lipase B (Novozym 435);
right, R. miehei lipase (Lipozyme IM)] in vacuo at 40, 60, and
80 °C for various periods.
lipase showed a steep increase in the extent of esteri-
fication with increasing molar ratio of oleic acid (17).
The time course of the formation of cholesteryl
myristate, cholesteryl stearate, and cholesteryl oleate
by Lipozyme IM-catalyzed transesterification of methyl
myristate, methyl stearate, and methyl oleate with
cholesterol (molar ratio 3:1, each) in vacuo at 80 °C for
various periods is shown in Figure 5. It is evident that
transesterification with methyl oleate is slightly pre-
ferred as compared to the corresponding reactions with
saturated long-chain fatty acid esters such as methyl
myristate and methyl stearate. It is noteworthy that the
transesterification of methyl myristate and methyl
stearate with sitostanol, catalyzed by Lipozyme IM at
80 °C, leads by far to higher conversions than the
esterification reaction of saturated fatty acids with

5214 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Weber et al.
Ta ble 1. En zym e Activities of Va r iou s Lip a ses d u r in g Ester ifica tion a n d Tr a n sester ifica tion of Ster ols a n d Sta n ols in
Va cu o a t Differ en t Tem p er a tu r esa
fatty acid or
fatty acid ester
maximum
enzyme activityc
sterol or stanol
enzyme
temp (°C)
time (h)
conversionb (%)
(units‚g-1
)
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
sitostanol
5R-cholestan-3â-ol
cholesterol
cholesterol
cholesterol
cholesterol
lanosterole
papayad
oleic acid
methyl oleate
oleic acid
methyl oleate
oleic acid
methyl oleate
triolein
oleic acid
methyl oleate
oleic acid
methyl oleate
oleic acid
methyl oleate
triolein
oleic acid
methyl oleate
oleic acid
methyl oleate
oleic acid
methyl oleate
triolein
methyl oleate
methyl myristate
methyl stearate
methyl oleate
ethyl dihydrocinnamate
methyl oleate
40
40
40
40
40
40
40
60
60
60
60
60
60
60
80
80
80
80
80
80
80
80
80
80
80
80
80
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
24
48
24
48
48
48
96
96
6.1
10.1
13.6
11.2
8.6
0.08
0.06
0.17
0.08
0.08
0.08g
0.08
0.25
0.33
0.25
0.63f
0.4
1.0
0.5
0.42
0.83
0.92
1.8
papayad
Novozym 435
Novozym 435
Lipozyme IM
Lipozyme IM
Lipozyme IM
papayad
14.0
13.9
29.3
48.0
40.2
69.0
40.0
78.3
64.8
46.6
61.6
88.7
99.2
63.8
93.2
95.7
94.9
63.6
74.9
83.4
56.0
14.0
papayad
Novozym 435
Novozym 435
Lipozyme IM
Lipozyme IM
Lipozyme IM
papayad
papayad
Novozym 435
Novozym 435
Lipozyme IM
Lipozyme IM
Lipozyme IM
Lipozyme IM
Lipozyme IM
Lipozyme IM
Lipozyme IM
Lipozyme IM
Lipozyme IM
1.3
3.2
2.3
3.1
1.1g
0.96g
0.96g
0.19f
0.02f
a
Experimental conditions: 100 µmol of sterol; molar ratio of sterol/fatty acid and sterol/fatty acid methyl ester, 1:3; molar ratio of
sterol/trioleoylglycerol 2:3; amount of various lipases, 50 mg; 20-40 mbar. Determined by GC. c Enzyme units were calculated as 1
b
µmol of steryl ester formed‚min-1‚g-1 lipase from the initial rates (4 h) of esterification or transesterification as described under
d
Experimental Procedures. A fine powder obtained from crude granular papaya latex preparation by grinding in a mortar was used (cf.
Experimental Procedures). e Commercial lanosterol preparation contained around 70% lanosterol and 30% dihydrolanosterol. The
conversions were calculated as total amount of steryl esters formed. f Twenty-four-hour value was used for calculation of enzyme activity.
g
Eight-hour value was used for calculation of enzyme activity.
F igu r e 4. Time course of the formation of sitostanyl oleate
by transesterification of sitostanol with methyl oleate at
various molar ratios (], 1:1; O, 1:3; 0, 1:5), catalyzed by R.
miehei lipase (Lipozyme IM, 50 mg/100 µmol of sitostanol) in
vacuo at 80 °C for various periods.
acids and relatively low temperature used in the latter
reaction.
Figure 6 summarizes the data on the formation of
steryl oleates by Lipozyme IM-catalyzed transesterifi-
cation of methyl oleate with sets of equimolar mixtures
of (A) 5R-cholestan-3â-ol and cholesterol, (B) sitostanol
and cholesterol, and (C) 5R-cholestan-3â-ol and sito-
stanol (molar ratio sterols/methyl oleate ) 1:3). It is
obvious from these experiments under competitive
conditions that saturated stanols such as 5R-cholestan-
3â-ol and sitostanol are the preferred substrates as
compared to the ∆5-unsaturated cholesterol in transes-
terification reaction with methyl oleate using Lipozyme
IM as catalyst. In contrast, the enzyme activity of Cd.
rugosa lipase was found to be higher in the esterification
reaction of ∆5-unsaturated cholesterol with oleic acid as
F igu r e 3. Effects of acyl donor and temperature on the time
course of formation of sitostanyl oleate by esterification of 100
µmol of sitostanol with 300 µmol of oleic acid (b) and by
transesterification of 100 µmol of sitostanol with 300 µmol of
methyl oleate ([) or 150 µmol of triolein (9) catalyzed by 50
mg of R. miehei lipase (Lipozyme IM) in vacuo at 40, 60, and
80 °C for various periods.
cholesterol using Cd. rugosa lipase at 40 °C (17), which
we attribute to the high melting points of these fatty

Lipase-Catalyzed Steryl Ester Preparation
J. Agric. Food Chem., Vol. 49, No. 11, 2001 5215
Figu r e 5. Time course of the formation of cholesteryl myristate,
cholesteryl stearate, and cholesteryl oleate via transesterifi-
cation of methyl myristate (0), methyl stearate (O), and methyl
oleate (]) with cholesterol (molar ratio 3:1, each), catalyzed
by R. miehei lipase (Lipozyme IM, 50 mg/100 µmol cholesterol)
in vacuo at 80 °C for various periods.
F igu r e 7. Time course of the formation of cholesteryl dihy-
drocinnamate ([) and lanosteryl oleate (9) via transesterifi-
cation of ethyl dihydrocinnamate with cholesterol (molar ratio
∼10:1) and of methyl oleate with lanosterol (molar ratio 6:1),
respectively, catalyzed by 50 mg of R. miehei lipase (Lipozyme
IM) in vacuo at 80 °C for various periods.
(14%, cf. Table 1) only after prolonged time (96 h) using
a large excess (molar ratio 6:1) of methyl oleate; at lower
ratios of methyl oleate to lanosterol the conversions were
substantially lower (data not shown). The results given
in Figure 7 demonstrate that the extent of transesteri-
fication of cholesterol with ethyl dihydrocinnamate and
of lanosterol with methyl oleate using Lipozyme IM as
biocatalyst under the above conditions is rather low,
which we attribute to the bulky structure of the dihy-
drocinnamoyl moiety and sterical hindrance by the 4,4-
dimethyl substituents of lanosterol.
Cholesterol was also transesterified with 1,8-octanedi-
carboxylic acid dimethyl ester using Lipozyme IM as
catalyst in vacuo (molar ratio of 1:4) for a prolonged time
(72 h) to give 1,8-octanedicarboxylic acid methylcholes-
teryl diester in moderate yield (75%) and 1,8-octanedi-
carboxylic acid dicholesteryl ester in poor yield (5%; data
not shown). Moreover, ethylcholesteryl carbonate and
oleylcholesteryl carbonate (Figure 1) were formed in low
yield (∼20%, each) by transesterification of cholesterol
with diethyl carbonate (molar ratio around 1:40; mo-
lecular sieve, 4 Å; 400 mbar; 60 °C; 48 h) and of eth-
ylcholesteryl carbonate with oleyl alcohol (molar ratio
of 1:3; 20 mbar; 80 °C; 48 h) using Lipozyme IM as
catalyst (data not shown). These cholesterol derivatives
having liquid crystalline properties may be used in
liquid crystal devices.
Figure 8 shows the effect of repeated use of the lipase
catalyst on the extent of transesterification of sitostanol
with methyl oleate, catalyzed by papaya lipase, No-
vozym 435, and Lipozyme in vacuo. It is evident that
both papaya lipase and Novozyme 435 lost some activity
upon repeated use; however, between 20 and 40% of
their activity was retained even after 10 repeated uses.
Lipozyme IM retained most of its activity even after 10
repeated transesterification reactions.
Table 1 summarizes the data on maximum conversion
and enzyme activity in esterification and transesterifi-
cation reactions of sitostanol, 5R-cholestan-3â-ol, and
cholesterol with different acyl donors, catalyzed by
papaya lipase, Novozym 435, and Lipozyme IM in vacuo
at various temperatures. From these results it is obvious
that sitostanol was converted in high to near-quantita-
tive yields to the corresponding long-chain acyl esters
via esterification with oleic acid or transesterification
with methyl oleate or trioleoylglycerol using Lipozyme
IM and Novozym 435 as biocatalysts in vacuo (20-40
mbar) at 80 °C, whereas the conversion was markedly
F igu r e 6. Time course of the formation of (A) 5R-cholestan-
3â-yl oleate ([) and cholesteryl oleate (O), (B) sitostanyl oleate
(9) and cholesteryl oleate (O), and (C) 5R-cholestan-3â-yl oleate
([) and sitostanyl oleate (9) via transesterification of 300 µmol
of methyl oleate with 100 µmol, each, of equimolar mixtures
of (A) 5R-cholestan-3â-ol and cholesterol, (B) sitostanol and
cholesterol, and (C) 5R-cholestan-3â-ol and sitostanol, cata-
lyzed by 50 mg of R. miehei lipase (Lipozyme IM) in vacuo at
80 °C under competitive conditions.
compared to the esterification of saturated sitostanol
with the same fatty acid (17).
Figure 7 shows the time course of the formation of
cholesteryl dihydrocinnamate (Figure 1) by Lipozyme
IM-catalyzed transesterification of ethyl dihydrocin-
namate with cholesterol (molar ratio 15:1) in vacuo at
80 °C for various periods. These results show that
cholesteryl dihydrocinnamate is formed in moderate
yield (56%, cf. Table 1) only after a prolonged time (96
h) using a large excess of ethyl dihydrocinnamate; at
lower ratios of ethyl dihydrocinnamate to cholesterol the
conversions were substantially lower (data not shown).
Cholesteryl dihydrocinnamate may be used in liquid
crystal devices or as an oil-soluble UV-B filter in sun-
care and cosmetic formulations.
The time course of the transesterification of methyl
oleate with lanosterol, catalyzed by Lipozyme IM in
vacuo at 80 °C for various periods (Figure 7), demon-
strates that lanosteryl oleate is formed in low yield

5216 J. Agric. Food Chem., Vol. 49, No. 11, 2001
Weber et al.
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F igu r e 8. Percent conversion of sitostanol to sitostanyl oleate
in the transesterification of sitostanol (100 µmol) with methyl
oleate (300 µmol), catalyzed by papaya (Cr. papaya) latex
lipase (50 mg), Cd. antarctica lipase B (Novozym 435, 50 mg),
or R. miehei lipase (Lipozyme IM, 50 mg) after repeated use
of the lipase catalyst as described under Experimental Pro-
cedures. The reactions were carried out in vacuo at 80 °C for
24 h.
lower at 60 °C and was very low at 40 °C. Conversions
observed with papaya lipase were generally lower at all
temperatures than those with Novozym 435 and Li-
pozyme IM. Highest conversion rates were observed in
transesterification reactions of sterols and stanols with
methyl oleate or triolein at 80 °C using Lipozyme IM
as catalyst. When Lipozyme IM was used as catalyst,
transesterification of cholesterol with ethyl dihydrocin-
namate gave a moderate yield of cholesteryl dihydro-
cinnamate after prolonged reaction time using a large
excess of ethyl dihydrocinnamate, whereas transesteri-
fication of lanosterol with methyl oleate resulted in very
low yield of lanosteryl oleate.
(17) Weber, N.; Weitkamp, P.; Mukherjee, K. D. Fatty acid
steryl, stanyl and steroid esters by esterification and
transesterification in vacuo using Candida rugosa lipase
as catalyst. J . Agric. Food Chem. 2001, 49, 67-71.
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tions with lipases. In Biotechnology, 2nd ed.; Rehm, H.-
J ., Reed, G., Eds.; Wiley-VCH: Weinheim, Germany,
1998; Vol. 8a: Biotransformations, pp 37-191.
It is interesting to note that enzyme activity of Cd.
rugosa lipase in the esterification reaction of sitostanol
with oleic acid yielding sitostanyl oleate is by far higher
(∼25 units‚g-1) (17) than the enzyme activity of Li-
pozyme IM or Novozym 435 in the transesterification
reaction of sitostanol with methyl oleate (∼2-3 units‚g-1
)
(Table 1); similar differences were obtained in the
reaction of cholesterol with oleic acid and methyl oleate,
respectively (∼32 vs ∼1 unit‚g-1; Table 1) (17).
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Received for review J une 5, 2001. Revised manuscript received
September 11, 2001. Accepted September 11, 2001.
J F0107407