J Am Oil Chem Soc (2008) 85:47–53
Umbelliferyl Assay
49
1,3-diolein was performed by TLC (chloroform:acetone
96:4 v/v), Iatroscan (conditions see above) and NMR-
spectroscopy (1H-NMR (300 MHz, CHCl3), d 0.88 (t, 6 H,
J = 7.0 Hz, CH3), d 1.26–1.30 (m, 40 H, CH2 group of
fatty acid chain length), d 1.63 (t, 4 H, J = 7.3 Hz,
COOCH2CH2), d 2.02 (m, 8 H, CHCH2), d 2.34 (t, 4H,
J = 7.7 Hz, COOCH2), d 4.07–4.20 (5 H, glycerol back-
bone), d 5.34 (m, 4 H, CH)).
The temperature optimum of lipases was determined using
4-methylumbelliferyl butyrate (4-MUB) in a microtiterplate
(MTP) assay. Lipase hydrolysis yields the fluorescent
compound 4-methylumbelliferone (4-MU), which was
quantified using an excitation wavelength of 355 nm and an
emission wavelength of 460 nm. Enzyme samples were
incubated in an Eppendorf thermoshaker at 65 °C for 90 s or
at 85 °C for 30 s and then stored on ice prior to fluorimetric
measurement. 150 ll of these treated enzyme solutions
(10 mg dissolved in 1 ml milk) were transferred to the wells
of an MTP and mixed with 150 ll substrate (final concen-
tration 90 lM 4-MUB in phosphate buffer pH 5.7, 50 mM)
using the pump in the fluorimeter (Galaxy-Fluostar, BMG,
Offenburg, Germany). The pH of this milk-buffer solution
was adjusted to pH 6.0, the temperature was set to 37 °C,
and the plate was shaken between the measurement cycles.
Lipase Immobilization
Selected Verenium enzymes were immobilized by
adsorption on Celite 545. For this, 50 mg lipase were
dissolved in 1.5 ml sodium phosphate buffer (pH 7.5,
50 mM) and afterwards mixed with 500 mg support. The
solution was stirred for 15 min. Chilled acetone was added
in small portions and the preparation was collected by
filtration. After several washing steps with acetone, the
filtrate was dried overnight under vacuum.
Alcoholysis of Triolein and Synthesis of 1,3-Diolein
Synthesis of 1,3-Dioleyl-2-palmitoyl-sn-glycerol
The reaction was performed according to the method of
Soumanou and Bornscheuer [10]. Triolein (20 mmol),
ethanol (20 mmol) and 10% (w/w) immobilized RMIM
were mixed in a 50-ml glass flask. The reaction mixture was
incubated in a water bath at 40 °C and agitated with a
magnetic stirrer at 200 rpm. After 5 and 10 h, 20 mmol
ethanol were added. Reaction progress was monitored using
thin layer chromatography (TLC) and Iatroscan analysis.
After 24 h, the reaction was terminated by removal of
immobilized lipase. The desired ethyloleate was separated
on preparative scale by silica gel column chromatography
(petrolether:diethylether, 7:1, v/v) yielding 80% product.
The purity was checked using TLC (n-hexane:diethyl-
ether:acetic acid 87:13:0.2 v/v/v) and Iatroscan (benzene:
chloroform:acetic acid, 50:30:0.5, v/v/v).
For this, purified 1,3-diolein (54 lmol) and vinylpalmitate
(80 lmol) were dissolved in 1 ml n-hexane in 1.5 ml glass
vials. All reaction components were adjusted before the
reaction to a fixed water activity (aw = 0.11) using satu-
rated salt solution [12]. Then activated molecular sieves
˚
(3 A) and 20 mg immobilized enzyme were added and the
reaction mixture was incubated at 40 °C and 1,000 rpm.
The reaction progress was monitored by TLC (n-hex-
ane:diethylether:acetic acid, 70:30:1, v/v/v) and Iatroscan
(benzene:chloroform:acetic acid, 50:30:0.5, v/v/v) analysis.
HPLC Separation of Triacylglycerols
The 1,3-diolein was produced according to a published
procedure [11]. Glycerol (12 mmol) was mixed with 1 g
silica gel in a 50 ml glass flask until the mixture became
dry. The purified ethyloleate (24 mmol), 20 ml tert-butyl-
methyl-ether (MTBE) and 0.5 g immobilized RMIM were
then added. The ethanol produced was removed from
The composition of the structured triglycerides was deter-
mined by HPLC on a Chromspher 5 Lipids column
(250 9 4.6 mm, Varian, Darmstadt, Germany) with an
evaporative light-scattering detector (65 °C, 1.5 ml/min
nitrogen) at a column temperature of 40 °C and a flow rate
of 1 ml/min using 0.5% acetonitrile in n-hexane as solvents
[13].
˚
the reaction mixture by activated molecular sieve (4 A).
All reaction compounds were adjusted to a defined water
activity (aw = 0.11) using saturated salt solutions overnight
prior to the reaction [12]. The reaction progress was
monitored by TLC and Iatroscan analysis. The purification
of the regioisomerically pure 1,3-diolein was carried out
using silica gel column chromatography. In the first sepa-
ration step, an n-hexane:diethylether gradient system (5:1-
1:1 v/v) was used, followed by a chloroform:acetone
(96:4 v/v) eluent mixture. The identification of the desired
Results and Discussion
Preparation of FAME and Gas Chromatographic
Analysis
The chosen method to monitor lipase selectivity was con-
version of released FA to FAME, followed by quantitation
123