ferent organ-specific and wound-regulated expression patterns, J. [42] Kohlmann, M., A. Kuntzsch, C. Wasternack, and I.
Feussner,
Biol. Chem. 271 (1996), 21012–21019.
[34] Feussner, I., Are lipoxygenases implicated in the degradation of
membrane constituents of chloroplasts during stress? Biol. Chem.
378 (1997), S163.
[35] Feussner, I., A. Böhm, B. Parthier, and C. Wasternack, Are lipoxy-
genases implicated in the degradation of membrane constituents of
chloroplasts during methyl jasmonate-induced senescence? Eur. J.
Cell Biol. 72 Suppl. 43 (1997), 95.
[36] Hause, B., U. zur Nieden, J. Lehmann, C. Wasternack, and B.
Parthier, Intracellular localization of jasmonate-induced proteins
in barley leaves, Bot. Acta 107 (1994), 333–341.
[37] Parthier, B., Jasmonates: hormonal regulators or stress factors in
leaf senescence?, J. Plant Growth Regul. 9 (1990), 57–63.
[38] Prakash, T. R., P. M. Swamy, P. Suguna, and P. Reddanna, Charac-
terization and behaviour of 15-lipoxygenase during peanut cotyle-
donary senescence, Biochem. Biophys. Res. Commun. 172
(1990), 462–470.
[39] Gut, H., and P. Matile, Breakdown of galactolipids in senescent
barley leaves, Bot. Acta 102 (1989), 31–36.
[40] Holtman, W. L., J. C. Vredenbregt-Heistek, N. E. Schmitt, and I.
Feussner, Lipoxygenase-2 oxygenates storage lipids in embryos of
germinating barley, Eur. J. Biochem. 248 (1997), 452–458.
[41] Avdiushko, S., K. P. C. Croft, G. C. Brown, D. M. Jackson, T. R.
Hamilton-Kemp, and D. Hildebrand, Effect of volatile methyl
jasmonate on the oxylipin pathway in tobacco, cucumber, and
Arabidopsis, Plant Physiol. 109 (1995), 1227– 1230.
Einsatz von Enzymen in und für die Verbraucher-Endprodukte/
Application of enzymes in and for consumer-ready products
Application of phospholipases in the edible oil industry
Microbial hydrolytic enzymes with phospholipase activites were found
in Aspergillus strains, suitable for the hydrolysis of phospholipids in
soybean, rapeseed, and sunflower oil during the enzymatic degumming
of edible oils. The microbial enzyme is significantly different from
pancreatic phospholipase. Calcium is not essential, but enhances the
activity.
1 Introduction
Phospholipases used for industrial processes in the food
industry are mainly produced from porcine or bovine
pancreas. The microbially produced phospholipase D was
mainly discussed for use in the synthesis of chemicals [1, 2]
and phospholipase A1 for the removal of free fatty acids from
oil preparations [3]. Phospholipases C and D were chiefly
* Röhm Enzyme GmbH, Department FEA, 64293 Darmstadt, Germany
152 Fett/Lipid 100 (1998), Nr. 4-5, S. 152–156 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
Effect of jasmonic acid methyl ester on enzymes of the lipoxy-
genase pathway in barley leaves, in: Natural Product Analysis.
(Ed. Schreier, P.), in press.
[43] Feussner, I., and H. Kühn, The lipid body lipoxygenase from cu-
cumber seedlings exhibits unusal reaction specificity, FEBS Lett.
367 (1995), 12–14.
[44] Böhm, A., C. Wasternack, and I. Feussner, Lipoxygenase catalyzed
oxygenation of complex lipids, Biol. Chem. 378 (1997), 163.
[45] Gardner, H. W., Soybean lipoxygenase-1 enzymatically forms
both 9(S)- and 13(S)-hydroperoxides from linoleic acid by a
pH-dependent mechanism, Biochim. Biophys. Acta 1001 (1989),
274–281.
[46] Zhang, L. Y., and M. Hamberg, Specificity of two lipoxygenases
from rice: unusual regiospecificity of a lipoxygenase isoenzyme,
Lipids 31 (1996), 803–809.
[47] Hamberg, M., and B. Samuelsson, On the specificity of the oxyge-
nation of unsaturated fatty acids catalyzed by soybean lipoxidase,
J. Biol. Chem. 242 (1967), 5329–5335.
Address of the authors: Dr. Ivo Feussner (corresponding author) and
PD Dr. C. Wasternack, Institut für Pflanzenbiochemie, Weinberg 3,
06120 Halle/Saale, Germany.
[Received: November 20, 1997; accepted: January 13, 1998].
Bruno H. Winter*, Kornelia Titze* und
Volker Marschner*
Einsatz von Phospholipasen in der Lebensmittelindustrie.
Hydrolytisch spaltende Enzyme mit Phospholipaseaktivität wurden im
Kulturüberstand von Aspergillus entdeckt. Diese Enzyme eignen sich
für die Hydrolyse der Phospholipide in Soja-, Raps- und Sonnenblu-
menöl während des enzymatisch katalysierten Entschleimungsschrittes
bei der Herstellung von Speiseölen. Das mikrobielle Enzym unterschei-
det sich signifikant von der Phospholipase aus Schweinepankreas. Es
benötigt kein Calcium für seine Aktivität. Diese wird aber durch den
Zusatz an Calcium gesteigert.
used for medical and analytical purposes [4]. For the enzy-
matic degumming of edible oils, phospholipases of the A2-
type were reported to be suitable [5]. Other applications of
phospholipase A2 include the synthesis of triglycerides
enriched in polyunsaturated fatty acids (PUFAs) from soy-
bean lysolecithin [6]. Enzymatic degumming of crude edible
oils reduces the amounts of acid, base, and waste during the
refining process [5]. It also allows the extraction of lecithin
and lysolecithin as a valuable byproduct for the fortification
of other foods. Microbially produced enzymes therefore not
0931-5985/98/0405-0152$17.50+.50/0
only yield in a more environmentally friendly production
process, but also provide a product, which can be sold in
Islamic countries and in the Jewish community.
Many microbially produced phospholipases A1 and D
are known, mainly from Streptomyces [7, 8], Aspergillus,
and Bacillus species. In 1994, a Streptomyces violaceoruber
phospholipase A2 was patented by Suzuki and Sugiyama [9].
In 1991, production of porcine pancreas phospholipase by
Saccharomyces cerevisiae was achieved by Bekkers et al.
[10], at yields, which were too low for an economical indus-
trial process.
2 Materials and Methods
2.1 Strains
All Aspergillus strains came from the strain collection of
Röhm Enzyme GmbH, Darmstadt. The work presented in this
paper was performed with an enzyme preparation produced
by Aspergillus sp. RH 3046.
2.2 Chemicals
Epicuron-200 (No. 139 029, purified phosphatidyl choline
from soybean) was purchased from Lucas Meyer, Hamburg,
Germany. The test kit for the determination of free fatty acids
(No. 1383175) was from Boehringer Mannheim,
Germany. Crude rapeseed and soybean oil were a gift from
Lurgi Öl Gas Chemie GmbH, Frankfurt, Germany. All other
chemicals were of analytical grade, from either Merck or
Sigma.
2.3 Methods
2.3.1 Phospholipase activity
Phospholipase activity was determined with a pH-stat
(Metrohm) according to QS analytical procedure QA
1/011/029 of Röhm Enzyme GmbH. One unit of phospho-
lipase corresponds to the amount of enzyme, which releases
1 mmol of fatty acids per minute under described conditions.
Substrate solution: 1 g Epicuron-200, 100 ml deionized
water, and 5 ml 0.32 M CaCl2 solution were homogenized
with an Ultra-Turrax for 2 min at 24,000 rpm. This substrate
solution may be kept for 3–4 d at 4° C. The assay was
performed by mixing 10 ml substrate solution with 10 ml 1%
Triton X-100 solution and 5 ml 3.3 mM citric acid monohy-
drate solution in a wide-shaft Erlenmeyer flask with a
magnetic stirring bar and incubated for 10 min at 40° C. After
addition of 0.1 ml of appropriately diluted enzyme solution
(< 2.5 PLU* g–1) the reaction mixture was incubated for
10 min at 40° C. At the end of the incubation period the reac-
tion mixture was titrated with 10 mM KOH to pH 10.0, the
first 5 ml KOH being added quickly (within approximately
1 min). Consumption of KOH was monitored. Blanks were
measured with a heat-inactivated enzyme sample, for which
an enzyme stock solution was kept at 95° C for 15 min. After
cooling to ambient temperature the solution was used as
described for the active enzyme sample.
2.3.2 FIP lipase activity
FIP (International Pharmaceutical Federation) lipase
activity was measured according to the phospholipase
* PLU = Phospholipase units.
Fett/Lipid 100 (1998), Nr. 4-5, S. 152–156
method, but using 33 mg ml–1 olive oil as substrate, 11.6 mg
ml–1 sodium taurodeoxycholate, and 30 mg ml–1 gum arabic
in the assay mixture.
2.3.3 Influence of metal ions and EDTA
For the determination of the influence of metal ions
and EDTA, the liberated fatty acids were measured with a
Boehringer test kit (Free fatty acids No. 1383175). With the
exception of the assays with ETDA addition, Ca2+ was
always added to the assay.
2.3.4 Effect of temperature and pH on enzyme stability
and activity
The effect of pH on the enzyme activity was determined
with the Boehringer free fatty assay kit at 20 and 55° C using
Epicuron-200 as substrate. Temperature optimum was deter-
mined with the pH-stat method after incubating diluted
enzyme samples for 1 h at the appropriate temperature at
pH 4.0. The effect of temperature on enzyme stability was
determined by pre-incubating the diluted enzyme samples at
pH 4.0 in a premix for the assay, without the substrate solu-
tion, at 50–65° C for various periods of time. The activity
was measured with the pH-stat method.
2.3.5 Free fatty acid concentration in oil samples
Free fatty acids in the oil samples from the degumming
experiments were determined according to DGF-Einheits-
methode C-V 2 (57).
2.3.6 Phospholipid concentration in oil samples
Phospholipid concentration in the oil samples was deter-
mined by adding 750 mg MgO to 0.5–2 g of an oil sample in
a quartz bowl, evaporating the residual water for 60 min at
120° C and ashing for 30 min at 850° C. The phosphorus
content of the ash was determined according to DGF-Ein-
heitsmethode C-VI 4 (61).
2.3.7 Enzymatic degumming experiments
Enzymatic treatment of crude oil samples was performed
on a lab-scale by continuously pumping the mixture con-
sisting of 500 units phospholipase per kg of oil, 0.2% citric
acid and 7% total water content, at pH 4.0 and at 55° C for
6 h with a rotary pump (Metabo, Germany, No. 27621) at a
speed of 500 rpm. Samples were taken every 90 min and
analyzed for free fatty acids and residual concentration of
phosphorous compounds in the oil phase. Heat inactivated
enzyme solution and water served as blanks.
Residual phosphorus concentrations in the oil phase from
lab-scale degumming experiments of rapeseed oil were
measured at the end of 6 h incubation periods. The enzyme
was added to oil of 55° C. The first sample was taken at the
end of the first batch after 6 h. The sludge was stored over-
night at 55° C, and was kept at 70° C for 1 h before being
reused in the second degumming batch for 6 h. The same
procedure was repeated once more. No fresh enzyme was
added to the second and third degumming batch.
2.4 Enzyme preparation
Aspergillus sp. RH 3046 was grown in a 30 l bioreactor
at 500 rpm, 0.5 vvm, 28° C, and controlled pH-conditions
(pH 4–8). The medium consisted of 3.75% maltodextrin, 3%
corn steep in liquor, and 0.5% (NH4)2HPO4. The supernatant
of the culture was used as enzyme source after concentration
by ultrafiltration (10 kDa cut-off), diafiltration, and sterile
filtration through Seitz EKS filter sheets.
153
3 Results and Discussion
3.1 Characterisation of the microbially produced
enzyme
3.1.1 pH and temperature optima of the microbial
enzyme dissolved in an oil in water emulsion
The microbial enzyme showed a very broad temperature
optimum between 40 and 60° C for a residual activity above
90% of the maximum activity as displayed in Fig. 1. The
temperature optimum includes the temperature interval
(55–60° C) at which the industrial process is run.
The pH optima of this enzyme are displayed in Fig. 2. At
room temperature the pH-optimum is very sharp at pH 4.5.
However, at 55° C, the temperature of the industrial process,
the enzyme exhibits a much broader pH optimum between
pH 3.5–4.0. A similar, but reversed phenomenon is known
for amyloglucosidase from Aspergillus niger, which shows a
much broader pH optimum at lower temperature (20° C) than
at higher temperature (60° C) [11]. Other phospholipases (A1
and A2) from Aspergillus niger were reported to have their
pH optimum at pH 4.5 [12]. However, the authors did not
measure the pH optimum at elevated temperatures, so that
it is not known, whether broadening of the pH optimum at
higher temperatures is a unique feature of the Aspergillus sp.
RH 3046 enzyme.
Fig. 1. Temperature optimum of the microbial enzyme with phospholi-
pase activity, measured in a water phase at pH 4.0.
Fig. 2. pH-optimum of the microbial enzyme at 20° C and 55° C,
measured in a water phase. The activity was measured at 20° C (filled
symbols) and 55° C (open circles).
154
Fig. 3. Temperature stability in water of enzyme samples from two
different fermentations. The fermentations were performed at low
pH values (pH 4) and high pH values (pH 7). Data represent incubations
at 50° C (pH 7, filled dot), 55° C (pH 7, triangle down), 60° C (pH 7,
filled square), and 60° C (pH 4, diamond).
3.1.2 Temperature stability in water solutions
The temperature stability of the enzyme was greatly in-
fluenced by the pH profile used during the fermentation pro-
cess of the strain. An example is displayed in Fig. 3. A shift
of the pH during fermentation to neutral pH conditions, as
they are normally not used for fungi, resulted in a more
thermolabile protein. The thermostability at 60° C of the
enzyme produced at pH 4 is similar to the enzyme stability at
50° C of the enzyme produced at pH 7. The temperature
stability curves shown were measured in water with citric
acid as buffer at pH 4.0. The data obtained by this method are
only valid for the given environment, as can be seen from the
data from application tests, given below.
3.1.3 Influence of metal ions and EDTA
The influence of EDTA, Cu2+, Fe2+/3+, Ni2+, and Zn2+ on
the enzyme activity was analyzed at 10 mM and 20 mM con-
centration, respectively. The results are shown in Tab. 1. In
contrast to pancreatic phospholipases, the microbial enzyme
does not depend upon Ca2+ for its activity, although removal
of Ca2+ by the addition of 20 mM EDTA resulted in a 50%
decrease in enzyme activity. Due to the fact that the enzyme
sample always contained some Ca2+ from the fermentation
medium, no effect was observed for the addition of 10 mM
EDTA. Of the tested metal ions, Fe3+ had a strong influence
on the enzyme activity. Fe2+ and Cu2+ also reduced the enzy-
matic activity to a significant degree. Fe2+ slowly oxidizes
to Fe3+ at pH 4 thereby enhancing its inhibitory effect. The
metal ion concentrations tested are far above those found in
Tab. 1. Influence of EDTA and metal ions on the activity of the micro-
bial phospholipase activity, using the standard assay conditions.
Added metal Residual Residual
ion or agent activity at activity at
10 mM [%] 20 mM [%]
Ca2+ 100.0 n.d.
Cu2+ 67.3 n.d.
Ni2+ 90.8 n.d.
Zn2+ 78.6 n.d.
Fe2+ 69.5 n.d.
Fe3+ 22.6 n.d.
EDTA 100.0 50.4
Fett/Lipid 100 (1998), Nr. 4-5, S. 152–156
the industrial process and only a minor influence on the
enzyme activity is expected during the industrial process.
The citric acid concentration present in the assay is similar to
that present in the industrial process, so that the results can
be used to estimate the effects to be expected in industrial
use.
3.2 Application in the degumming of oil
3.2.1 Degumming experiments – stability of enzyme
sample during prolonged use
Enzymatic degumming of crude edible oils on an indus-
trial-scale – EnzyMax process Dahlke et al. [5] – is per-
formed by producing a stable emulsion of crude oil with a
phosphorous content, composed of phospholipids of about
150–300 ppm P, citric acid, enzyme solution, and additional
water. This emulsion is stirred for 6 h at 55–60° C, during
which lecithin is enzymatically hydrolyzed to lysolecithin.
Lysolecithin is soluble in the water phase, whereas lecithin is
only soluble in the oil phase. The emulsion is broken in a
continuously operating separator, resulting in three streams:
oil, water, and sludge. The enzyme adheres to the sludge, and
the majority of the sludge is brought back into the process
stream, so that the enzyme can be reused several times. This
process was simulated in the lab. As the centrifuge is operat-
ed at higher temperatures, hold-up times at 70° C were in-
cluded.
According to stability data from enzyme samples in the
water phase, the enzyme should have been inactivated
towards the end of the first test cycle. However, the observed
decrease in enzyme activity was minimal. According to
earlier experiments, a minimum initial enzyme activity of
300 PLU kg–1 oil is necessary to decrease the phosphorous
content in the oil phase to a value below 10 ppm P, which was
still achieved with enzyme preparations, that had been used
for three times. The results thus indicate that the sludge sta-
bilizes the microbial enzyme and only minor amounts of
fresh enzyme need to be added continuously to the industrial
production process. Data shown in Tab. 2 were obtained for
the degumming of rapeseed oil. Degumming experiments
with soy oil showed very similar results.
3.2.2 Liberation of fatty acids from triglycerides
Treatment of crude oil with the microbial enzyme samples
resulted in an increase of free fatty acids in the oil phase.
Data of several experiments are shown in Tab. 2. For oils
with about 300 ppm P an increase of 0.2%–0.3% in free
fatty acids could be observed due to the hydrolysis of
lecithin to lysolecithin. Such values were obtained with pan-
creatic phospholipase. The values obtained with the micro-
bial enzyme preparation were greater than 0.3%, indicating
some additional lipase activity in the enzyme preparation.
Tab. 2. Residual phosphorus concentrations in the oil phase from
lab-scale degumming experiments of rapeseed oil at the end of 6 h in-
cubation periods with fresh and recycled enzyme.
Incubation Residual Free fatty Total period of time
period concentration acids at which enzyme
[ppm] P [%] was exposed to
elevated temperatures
of 55° C/70° C [h]
Initial conditions 244 2.55 0/0
End of 1st 6 3.81 6/0
End of 2nd 7 3.19 30/1
End of 3rd 8 3.04 60/2
Fett/Lipid 100 (1998), Nr. 4-5, S. 152–156
Measurements of lipase activity (FIP assay) in the microbial
enzyme sample with olive oil, however, showed no lipolytic
activity. Therefore, the microbial lipase might have some
kind of substrate specificity, either for shorter chain fatty
acids or for polyunsaturated fatty acids. Mustranta et al. [12]
determined a much higher phospholipase than lipase activity
(with olive oil) in their A. niger phospholipase A2 prepara-
tion. The results obtained with the Aspergillus sp. RH 3046
during the degumming of rapeseed oil also showed more
phospholipase than lipase activity. The spectrum of fatty
acids in olive oil and rapeseed oil is not identical. Further-
more, not all activity measurements were performed in the
same type of emulsion (oil in water or water in oil) or under
identical conditions (emulsifier, temperature), resulting in
different diffusion coefficients and solubility properties.
Therefore, these data cannot be directly compared.
So far, some microbial enzymes with phospholipase
activity are known. Phospholipase activity was also observed
as another activity of microbial lipases by Mustranta et al.
[12] and Jaeger et al. [13]. The ratio of lipolytic to phospho-
lipolytic activity varies widely between known lipases with
phospholipase activity. Pancreatic phospholipase, however,
is regarded as a true phospholipase without lipase activity.
The enzyme preparations used by Röhm Enzyme and by
Mustranta et al. [12] were technical enzyme preparations.
Therefore, one cannot conclude, whether enzyme mixtures
were present or whether the enzymes belong to that class of
microbial lipases, in which phospholipase activity predomi-
nates.
4 Conclusions
The microbial enzyme preparation from Aspergillus sp.
RH 3046 could be used in lab-scale experiments for the
degumming of crude edible oils, although the data for tem-
perature stability, as analyzed in a dilution in water/buffer,
showed no promising values. The enzyme does not depend
upon Ca2+ and some lipolytic activity could be observed
during the degumming of rapeseed oil. Both facts indicate
that the enzyme, if only one enzyme was present, might
belong to the class of lipases in which phospholipase activity
predominates. Further detailed studies with the purified
enzyme will be necessary to answer this question. The poten-
tial use of such microbial enzymes for industrial degumming
of edible oils will depend upon their price and on whether
fermentation and process conditions can be found, which
inhibit the lipase activity.
Acknowledgements
We thank Astrid Pfeifer and Gerhard Leiß for the production of the
enzyme samples.
References
[1] Buehler, M., and C. Wandrey, Oleochemicals by biochemical
reactions, Fat Sci. Technol. 94 (1992), 82.
[2] Vulfson, E. N., Enzymatic synthesis of surfactants, Surfactants-
Lipid-Chem. 118 (1992), 16.
[3] van Tilbeurgh, H., R. Bhikhabhai, L. G. Pettersson, and M.
Claeyssens, Separation of endo- and exo-type cellulases using a
new affinity chromatography method, FEBS 169 (1984), 215.
[4] Ruttloff H.: Industrielle Enzyme. Behr’s Verlag 1994.
[5] Dahlke, K., H. Buchold, E.-M. Münch, and B. Paulitz, First experi-
ences with enzymatic oil refining, Inform 6 (1995), 1284–1291.
[6] Heusch, R.: Emulsions, in: Ullmann’s Encyclopedia of Industrial
Chemistry. Eds. Gerhartz W., Y. S. Yamaoto, L. Kaudy, F. J. Roun-
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 saville, G. Schulz, VCH Verlagsgesellschaft mbH, Vol. A9 1987,
pp. 295–339.
[7] Sawada Haruji, Kudo Satoshi, Watanabe, Tsuneichi, Kuroda Akio,
Oki Takamasa, Jap. Pat. J58212783 (1983), Kyowa-Hakko.
[8] Jap. Pat. J63219373 (1988), Shokuhin-Sangyo-Bioreactor-Syst.
[9] Suzuki, Y., and M. Sugiyama, JP 6-327468 (1994), Asahi Chemical
Industry Co.
[10] Bekkers, A. C. A. P. A., P. A. Franken, C. J. van den Bergh, J. M. A.
Verbakel, H. M. Verheij, and G. H. Haas, The use of genetic
engineering to obtain efficient production of porcine pancreatic
phospholipase A2 by Saccharomyces cerevisiae, Biochimica et
Biophysica Acta 1089 (1991), 345.
[11] Uhlig, H.: Enzyme arbeiten für uns; Technische Enzyme und ihre
Anwendungen. Carl Hanser Verlag, München, Wien 1991.
Synthesis of structured triglycerides
by lipase catalysis
The synthesis of structured triglycerides containing medium chain
fatty acids (M) in sn1 and sn3-position and an unsaturated long-chain
fatty acid (L) in sn2-position of the glycerol backbone (MLM) with
1,3-regiospecific lipases has been investigated. In an one-step synthesis
(acidolysis or transesterification) various commercial lipases were
tested. In all reaction systems, the recovered product contained con-
siderable amounts of undesired byproduct (e.g. MLL). A significantly
higher concentration of MLM was obtained in a two-step process under
the control of the water activity: In the first step, the 2-monoglycerides
(2-MG) were prepared by alcoholysis (e.g. with ethanol) of pure triglyc-
erides (triolein and trilinolein) or a natural oil (cottonseed oil) with
lipase from Rhizopus delemar immobilized on Celite® in organic
solvents (e.g. methyl tert-butyl ether).The 2-MGs were purified by
crystallization and isolated in yields up to 71.8% (e.g. from triolein).
The 2-MGs obtained from cottonseed oil was esterified in the second
step with caprylic acid in n-hexane using lipase from Rhizomucor
miehei (Lipozyme). The final product contained more than 94%
caprylic acid in sn1- and sn3-position, whereas the sn2-position was
composed of 78% of unsaturated long-chain fatty acids.
1 Introduction
Enzyme-catalyzed modification of the structure of fats
and oils is of great interest. This process can be performed by
exploiting the regiospecificity of lipases in interesterification
of transesterification of fats and oils [1]. In such a reaction, it
is possible to incorporate a desired fatty acid on a specific
position of the triglyceride molecule, whereas the con-
ventional chemical process leads to a randomization of fatty
acids among the glycerol backbone [2]. Lipases with
1,3-positional specificity show interesterification activity in
two types of reactions in microaqueous organic media:
acidolysis reaction, in which free fatty acids react with a
triglyceride to generate a new triglyceride molecule, and
* Institut für Technische Biochemie, Universität Stuttgart, Stuttgart,
Germany.
156 Fett/Lipid 100 (1998), Nr. 4-5, S. 156–160 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
[12] Mustranta, A., P. Forssell, and K. Poutanen, Comparison of
lipases and phospholipases in the hydrolysis of phospholipids,
Process Biochemistry 30 (1995), 393.
[13] Jaeger, K.-E., and S. Wohlfarth, Bakterielle Lipasen: Bio-
chemie, Molekulargenetik und biotechnologische Bedeutung,
BioEngineering 9 (1993), 39.
Address of the authors: Dr. Bruno H. Winter, Kornelia Titze, Volker
Marschner, Röhm Enzyme GmbH, Department FEA, Kirschenallee 45,
64293 Darmstadt, Germany.
[Received: November 18, 1997; accepted: February 10, 1998].
Mohamed M. Soumanou*,
Uwe T. Bornscheuer*, Ulrike Schmid*,
and Rolf D. Schmid*
Synthese strukturierter Triglyceride durch Lipase-Katalyse. Die
Synthese von strukturierten Triglyceriden – die mittelkettige Fettsäuren
(M) in sn1- und sn3-Position und eine ungesättigte langkettige Fettsäu-
re (L) in sn2-Position am Glyceringerüst (MLM) tragen – durch 1,3-
regiospezifische Lipasen wurde untersucht. In einem Einschrittverfah-
ren (Acidolyse oder Umesterung) wurden verschiedene kommerzielle
Lipasen eingesetzt. In allen Reaktionssystemen wurden beträchtliche
Mengen des Nebenprodukts (z. B. MLL) gebildet. Wesentlich höhere
Gehalte an MLM wurden in einem Zweischrittverfahren bei kontrol-
lierter Wasseraktivität erzielt: Im ersten Schritt wurden 2-Monoglyceri-
de (2-MG) durch Alkoholyse (z. B. mit Ethanol) von reinen Triglyceri-
den (z. B. Triolein, Trilinolein) oder natürlichem Öl (Baumwollsaatöl)
mit Lipase aus Rhizopus delemar immobilisiert auf Celite® in organi-
schem Lösungsmittel (z. B. Methyl-tert-Butylether) dargestellt. Diese
2-MGs wurden durch Kristallisation gereinigt und in Ausbeuten bis zu
71,8% ausgehend von Triolein isoliert. Die 2-MGs aus Baumwoll-
saatöl wurden im zweiten Schritt mit Caprylsäure in n-Hexan mit Lipa-
se aus Rhizomucor miehei (Lipozyme) verestert. Das Endprodukt ent-
hielt mehr als 94% Caprylsäure in sn1- und sn3-Position, während an
der sn2-Position 78% ungesättigte langkettige Fettsäuren gebunden
waren.
transesterification reaction, in which two different triglycer-
ides react to yield new triglycerides. In both reactions, the
sn2-position of the reacting triglycerides remains unchanged
[3].
The nutritional value of triglycerides and their physico-
chemical properties are determined not only by the fatty acid
composition, but also on the positional distribution of the
acyl groups bonded to glycerol [4]. Structured triglyceride of
the MLM-type, which contain medium-chain fatty acids in
sn1- and sn3-position and long unsaturated fatty acids in
sn2-position have been reported as concentrated form of
calories. These triglycerides provide rapid delivery of energy
via oxidation of the more hydrophilic medium-chain fatty
acids, while at the same time providing an adequate supply
of essential fatty acids from the remaining 2-monoglyceride
[5]. Such triglycerides are extremely rare and are difficult to
prepare by chemical synthesis, due to many purification
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