68 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Weber et al.
pellets. 5-Pregnen-3â-ol-20-one and 5R-pregnan-3â-ol-20-one,
50 µmol each, were transesterified under similar experimental
conditions with 500 µmol of tripropionin in the presence of 100
mg of C. rugosa lipase preparation in vacuo at 35 °C. The
vacuum used was 20-40 mbar, measured at room tempera-
ture.
Esterification reactions with lipases from Ch. viscosum and
porcine pancreas were performed using 100 µmol of cholesterol,
300 µmol of oleic acid, and 50 mg of the respective enzyme
preparation for 24 h in vacuo at 40 °C. In the case of R.
arrhizus lipase, 50 µL of the enzyme preparation was dried in
vacuo at 40 °C before use.
P u r ifica tion of Ster yl a n d Sta n yl Ester s by Dea cid i-
fica tion . In a typical example, the reaction products (∼65 mg)
resulting from lipase-catalyzed esterification of oleic acid with
a stoichiometric amount of sitostanol were taken up in 6 mL
of diethyl ether and the enzyme catalyst was separated by
centrifugation. The supernatant was removed and extracted
three times with 3 mL, each, of 2% aqueous sodium carbonate
solution, followed by repeated extractions with water, to
remove the unesterified oleic acid as sodium salt. The diethyl
ether phase was dried over anhydrous sodium sulfate to yield
60 mg of a product containing 94% sitostanyl oleate, 5%
sitostanol, and <1% oleic acid.
P u r ifica tion of 5-P r egn en -20-on e-3â-yl P r op ion a te a n d
5r-P r egn a n -20-on e-3â-yl P r op ion a te. The reaction mix-
tures resulting from lipase-catalyzed transesterification of
tripropionin with 5-pregnen-3â-ol-20-one were taken up in 3
mL of diethyl ether, and the enzyme catalyst was separated
by centrifugation. The supernatant was removed, the solvent
was evaporated, and the reaction products (∼140 mg), dis-
solved in 6 mL of isohexane, were extracted three times with
3 mL, each, of methanol/water (95:5, v/v) to remove most of
the tripropionin. The isohexane phase was dried over anhy-
drous sodium sulfate, concentrated to a small volume, and
purified via silica gel chromatography, as described above, by
elution with 30 mL of isohexane/diethyl ether (4:1, v/v) to yield
8.4 mg of 5-pregnen-20-one-3â-yl propionate (45% of theory;
purity ) 93%). Similarly, 5R-pregnan-20-one-3â-yl propionate
was purified via extraction and silica gel chromatography as
described above.
Meltin g P oin ts. Melting points of steryl esters determined
with a Thermovar heating block (Reichert, Vienna, Austria)
were as follows: sitostanyl oleate, 44-45 °C; sitostanyl li-
noleate, <20 °C; cholesteryl stearate, 83-84 °C; cholesteryl
oleate, 48-50 °C; cholesteryl laurate, 92-93 °C; cholesteryl
butyrate, 110-111 °C; stigmasteryl oleate, 43-44 °C; ergo-
steryl oleate, 76-77 °C; 7-dehydrocholesteryl oleate, 69-70
°C; 5-pregnen-20-one-3â-yl propionate, 122-123 °C.
Th in -La yer Ch r om a togr a p h y (TLC). Aliquots were with-
drawn from the reaction mixtures, and the conversion was
checked by TLC on 0.3 mm layers of silica gel H (Merck) using
isohexane/diethyl ether (95:5, v/v); spots were located by iodine
staining. Alternatively, the TLC plates were sprayed with a
30% aqueous sulfuric acid and heated in an oven kept at 120
°C; ∆5-sterols and their esters appeared as red spots and
∆
5,7-sterols and their esters as blue spots (Liebermann-
Burchard test). The Rf values of the various compounds were
as follows: fatty acid steryl and stanyl esters, 0.6-0.7; fatty
acid methyl esters, 0.4-0.5; triacylglycerols, 0.3-0.35; sterols
and stanols, 0.1-0.15; unesterified fatty acids, <0.1.
For the separation of mixtures containing 5-pregnen-20-one-
3â-yl propionate, 5R-pregnan-20-one-3â-yl propionate, and
tripropionin, the TLC plates were developed with isohexane/
diethyl ether (80:20, v/v); Rf values were as follows: 5-pregnen-
20-one-3â-yl propionate and 5R-pregnan-20-one-3â-yl propi-
onate, 0.2-0.25; tripropionin, 0.15-0.2; 5-pregnen-3â-ol-20-
one and 5R-pregnan-3â-ol-20-one, <0.05.
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, unreacted sterols, stanols, or steroids as well as car-
boxylic acid steryl, stanyl, or steroid esters were analyzed
without derivatization 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 chro-
matograph 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 programming 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, and the injector
as well as flame ionization detector temperature was 350 °C.
Peaks in the gas chromatograms were assigned by comparison
of their retention times with those of commercially available
standards or those prepared by chemical or enzymatic syn-
thesis. Peak areas and percentages were calculated using
Hewlett-Packard 3365 series GC ChemStation software.
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. In a typical example, the reaction mix-
tures resulting from lipase-catalyzed esterification of oleic acid
with sitostanol were taken up in 3 mL of diethyl ether, and
the enzyme catalyst was separated by centrifugation. The
supernatant was removed, the solvent was evaporated, and
the reaction products (∼120 mg), dissolved in 1 mL of isohex-
ane/diethyl ether (9:1, v/v), were applied to a column (20 cm
× 1 cm i.d.) packed with silica gel as a slurry in isohexane.
Elution with 20 mL of isohexane/diethyl ether (95:5, v/v)
yielded 60.5 mg of sitostanyl oleate (88% of theory; purity
>95%). Subsequent elution with 20 mL of isohexane/diethyl
ether (1:1, v/v) yielded unreacted oleic acid together with
sitostanol.
RESULTS AND DISCUSSION
Lipase-catalyzed esterification and interesterification
reactions 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 acyl esters
of sterols, stanols, and steroids (Haraldsson, 1992;
Kazlauskas and Bornscheuer, 1998). We show here that
esterification and interesterification reactions, catalyzed
by C. rugosa lipase under vacuum, provide fatty acyl
esters of sterols and stanols in near-quantitative yields.
Similarly, short-chain acyl esters of sterols and steroids
are obtained in high yields by transesterification with
short-chain triacylglycerols, catalyzed by C. rugosa
lipase in vacuo at moderate temperature. Neither
organic solvent nor water is added to the reaction
mixtures, and no drying agent such as a molecular sieve
is used.
Figure 1 shows the time course of esterification of
sitostanol with oleic acid, catalyzed by various propor-
tions of C. rugosa lipase in vacuo at 40 and 60 °C. It is
evident that a near-quantitative conversion is achieved
at 40 °C. Figure 1 also shows that the extent of
conversions is lower at 60 °C than at 40 °C, which we
attribute to a partial deactivation of the lipase at the
higher temperature. It is also evident from Figure 1 that
at both temperatures the extent of conversion increases
with increasing proportion of the lipase; however, at 40
°C increasing the amount of lipase from 25 to 50 mg
does not lead to any further increase in the extent of
esterification. In all of the following enzymatic reactions,
if not stated otherwise, 50 mg of immobilized C. rugosa
lipase was used.