Two-enzyme system for the synthesis of 1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and 1-lauroyl-dihydroxyacetonephosphate

Article abstract of DOI:10.1016/S0009-3084(99)00123-1

A combination of two enzymes, phospholipase D (PL D) and C (PL C), was investigated for the production of two lysophospholipids, 1-lauroyl-rac- glycerophosphate (1-LGP) and 1-lauroyl-dihydroxyacetonephosphate (1-LDHAP). The high transphosphatidylation ability of phospholipase D from Streptomyces sp. allowed the formation of 1-lauroyl-phosphatidylglycerol (1-LPG) and 1- lauroyl-phosphatidyldihydroxyacetone (1-LPDHA) from phosphatidylcholine (PC) and 1-monolauroyl-rac-glycerol (1-MLG) and 1-lauroyl-dihydroxyacetone (1- MDHA), respectively. A two-phase system, diethyl ether/water, was chosen for the convenience of the recovery of the water insoluble products. A similar two-phase system was used for hydrolysis of the complex phospholipids by phospholipase C form Bacillus cereus, which released both lysophospholipids. Only trace amounts of phosphatidic acid (PA) were detected showing that the enzyme is highly selective for the release of the diacylglycerol and 1- lauroyl-rac-glycerophosphate and 1-lauroyl-dihydroxyacetonephosphate.

Full text of DOI:10.1016/S0009-3084(99)00123-1

Chemistry and Physics of Lipids
104 (2000) 175–184
www.elsevier.com/locate/chemphyslip
Two-enzyme system for the synthesis of
1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and
1-lauroyl-dihydroxyacetonephosphate
Carmen Virto *, Patrick Adlercreutz
Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund Uni6ersity, PO Box 124, S-221 00,
Lund, Sweden
Received 26 July 1999; received in revised form 4 October 1999; accepted 4 October 1999
Abstract
A combination of two enzymes, phospholipase D (PL D) and C (PL C), was investigated for the production of two
lysophospholipids, 1-lauroyl-rac-glycerophosphate (1-LGP) and 1-lauroyl-dihydroxyacetonephosphate (1-LDHAP).
The high transphosphatidylation ability of phospholipase D from Streptomyces sp. allowed the formation of
1-lauroyl-phosphatidylglycerol (1-LPG) and 1-lauroyl-phosphatidyldihydroxyacetone (1-LPDHA) from phosphatidyl-
choline (PC) and 1-monolauroyl-rac-glycerol (1-MLG) and 1-lauroyl-dihydroxyacetone (1-MDHA), respectively. A
two-phase system, diethyl ether/water, was chosen for the convenience of the recovery of the water insoluble products.
A similar two-phase system was used for hydrolysis of the complex phospholipids by phospholipase C form Bacillus
cereus, which released both lysophospholipids. Only trace amounts of phosphatidic acid (PA) were detected showing
that the enzyme is highly selective for the release of the diacylglycerol and 1-lauroyl-rac-glycerophosphate and
1-lauroyl-dihydroxyacetonephosphate. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Phospholipase D; Phospholipase C; Acyl-phosphatidylglycerol; Acyl-phosphatidyldihydroxyacetone; Acyl-rac-glyc-
erophosphate; Acyl-dihydroxyacetonephosphate
1. Introduction
choline and glycerol or dihydroxyacetone for the
synthesis of phosphatidylglycerol and phos-
phatidyldihydroxyacetone with PL D. A solvent/
aqueous two-phase system was found to be
convenient for the easy separation and recovery
of the water insoluble products. After purifica-
tion, the obtained phospholipids were treated with
a PL C from Bacillus cereus in a similar two-phase
system which rendered glycerophosphate and di-
hydroxyacetophosphate, this time as water-solu-
ble products.
The combination of phospholipases D and C
for the formation of phosphorylated organic wa-
ter-soluble molecules has been reported (Shinitzky
et al., 1993; Takami and Suzuki, 1994; D’Arrigo
et al., 1995). The authors used phosphatidyl-
* Corresponding author. Tel: +46-46-222-48-41; fax: +46-
46-222-47-13.
E-mail address: carmen.virto@biotek.lu.se (C. Virto)
0009-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(99)00123-1

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C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
The unique transphosphatidylation activity of
In this paper, we report the utilisation of a
phospholipase D from Streptomyces sp. for the
formation of a synthetic phospholipid (1-lauroyl-
phosphatidylglycerol, acyl-phosphatidylglycerol)
the phospholipase D has been extensively studied,
and their applications are multiple (for a review
see Servi, 1999). Plant phospholipases D, from
cabbage, castor bean or peanut seed, p.g., have
proven to be effective catalysts in transphos-
phatidylation reactions (Heller, 1978). However,
the simultaneous formation of the phosphatidic
acid by-product has focused the investigators on
the search for more selective enzymes on microor-
ganisms. The results have been various, showing
that there are enzymes possessing high transphos-
phatidylation activities and others with a solely
hydrolitic ability (Nakajima et al., 1994; Juneja et
al., 1988, 1989).
Phospholipase C is a very well studied enzyme
that catalyses the hydrolysis of the phosphoester
bond on phospholipids, such as phosphatidyl-
choline, into the phosphorylated polar head group
and the diglyceride. The applications of this en-
zyme are somewhat limited, since it does not have
a very broad selectivity and synthetic phospho-
lipids are poor substrates for the enzyme (Ries et
al., 1991; Servi, 1999). Ries et al. (1991) showed
that for optimal activity, the phospholipids must
contain an ester or ether bond in the sn 1 and an
ester bond in the sn 2 position of the glycerol
backbone and with a phosphocholine esterified in
the sn 3 position. Anything out of this pattern
resulted in a drop in the hydrolytic activity.
Enzymatic synthesis of lysophosphatidic acid
has been achieved using different aproaches, such
as the esterification of glycerophosphate and fatty
acid or fatty acid vinyl ester with lipases (Han and
Rhee, 1995; Virto et al., 1999). However, some
phosphatidic acid was also formed in most reac-
tion conditions. Lysophosphatidic acid can be
also synthesised with a combination of two en-
zymes. Thus, phospholipase A2 (PL A2) can be
used for the hydrolysis of phosphatidylcholine to
form lysophosphatidylcholine, which can be fur-
ther hydrolysed by PL D to give lysophosphatidic
acid (Long et al., 1967). If natural PC is used,
LPA with different fatty acid composition is
formed. Van der Bend et al. (1992) reported the
synthesis of 1-oleoyl-LPA via phosphorylation of
dioleoylglycerol with diacylglycerol kinase form-
ing phosphatidic acid, which was then hydrolysed
with PL A2.
and
a
novel phospholipid (1-lauroyl-phos-
phatidyldihydroxyacetone, acyl-phosphatidyldihy-
droxyacetone). The enzyme has proven to be very
selective for the transphosphatidylation reaction.
The nuclophile 1-monolauroyl-rac-glycerol ap-
peared to be preferred to the ketone counterpart.
Acyl-phosphaditylglycerol has been shown to ex-
ist as a minor component of the phospholipids in
microorganism membranes (Makula et al., 1978;
De Siervo and Homola, 1980; Beach et al., 1991).
Furthermore, in lysosomes together with bis
(monoacylglycerol) phosphate it participates in
the turnover of fatty acids in the metabolism of
complex phospholipids (Waite et al., 1990;
Huterer and Wherret, 1990). The physical and
chemical properties of these phospholipids should
be of fundamental, and may be of practical,
interest.
The phospholipids were readily hydrolysed by a
phospholipase C form B. cereus giving 1-lauroyl-
rac-glycerophosphate (lysophosphatidic acid) or
1-lauroyl-dihydroxyacetonephosphate and diacyl-
glycerol. The enzymatic rates were similar sug-
gesting that the hydroxyl or ketone group in the
sn 2 position is of minor relevance. However, the
enzyme appeared to be somewhat more selective
for the hydrolysis of the 1-lauroyl-phosphatidyl-
glycerol, since the formation of phosphatidic acid
from the hydrolysis of the terminal phosphoester
bond on the molecule was lower than with 1-lau-
royl-phosphatidyldihydroxyacetone.
Increasing
amounts of phosphatidic acid in the media did
not inhibit the hydrolytic reaction.
2. Materials and methods
2.1. Materials
Phospholipase D from Streptomyces sp. and
phospholipase C from B. cereus, phosphatidyl-
choline (\99%) and N-(2-Hydroxyethylpiper-
azine-N%-(2-ethanesulfonic acid) (HEPES), were
products from Sigma Chemical Co. (St. Louis,

C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
177
MO). Monolaurin was purchased from Larodan
AB (Malmo¨, Sweden), phosphatidic acid and
lysophosphatidic acid were obtained by the action
of phospholipase D from cabbage towards phos-
phatidylcholine and lysophosphatidylcholine, re-
spectively. Diacylglycerol was obtained by the
action of PL C on phosphatidylcholine. Sodium
acetate trihydrate, copper (II) sulphate pentahy-
drate, calcium chloride dihydrate, silica gel 60 and
all the solvents were purchased from Merck
(Darmstadt, Germany) and were of analytical
grade. 1-lauroyl-dihydroxyacetone was produced
in our laboratory (Virto et al., 1999).
shaker. The reactions were started by the addition
of 4 units of phospholipase D. After the comple-
tion of the reaction the mixtures were centrifuged
at 6000 r.p.m. The upper ether phase, containing
products, was collected and the solvent evapo-
rated in a rotavapor. If no phosphatidic acid was
present, the products were separated from the
1-monolauroyl-rac-glycerol or 1-monolauroyl-di-
hydroxyacetone by simple precipitation with ace-
tone. For the purification of reactions containing
phosphatidic acid the mixtures were dissolved on
chloroform and separated by chromatography on
silica gel. The solution was applied to the column
and eluted with chloroform until no more 1-MLG
or 1-MLDHA was detected in the eluate. 1-LPG
and 1-LPDHA were then eluted with a mixture of
chloroform:methanol:NH3 (70:21:2). Under these
conditions phosphatidic acid is retained on the
column.
2.2. Methods
2.2.1. Phospholipase D catalysed reactions
Typically, phosphatidylcholine to a concentra-
tion of 10 mM and different concentrations of
1-monolauroyl-rac-glycerol or 1-monolauroyl-di-
hydroxyacetone (from 5 to 200 mM) were dis-
solved in 0.4 ml diethyl ether. Added to the
solution were 0.4 ml Na-acetate buffer (0.2 M),
pH 5.5 containing 40 mM CaCl2, with the subse-
quent formation of a two-phase system. The reac-
tion media were incubated in an orbital shaker
(100 r.p.m.) at 25°C. The reactions were started
by the addition of 0.1 units PL D. Samples of 10
ml were withdrawn from the ether phase at inter-
vals and the solvent was allowed to evaporate
before the addition of 25 ml tetrahydrofuran. For
the final quantification of the products, the re-
maining reaction mixture was mixed with 50 ml of
1 N HCL and 500 ml of chlorofrom:methanol
(2:1). The mixtures were thoroughly vortexed and
centrifuged at 6000 r.p.m for 10 min. The lower
chloroform rich part was collected for analysis.
No phospholipids were detected in the upper
methanolic phase which was discharged. Obtained
samples were then analysed by HPTLC.
2.2.3. Fatty acid analysis of 1-LPG and
1-LPDHA
For the verification of the structure the prod-
ucts were subjected to fatty acid analysis
(Svensson et al., 1992). Bands on TLC plates
containing 1-LPG and 1-LPDHA, and their prod-
ucts after hydrolysis with phospholipase C, were
scraped and mixed with 2 ml of 0.5 M sodium
methoxide in methanol. After incubation for 10
min at 50°C, 200 ml of hexane and 4 ml of
saturated NaCl solution were added. The mixture
was vortexed and centrifuged at 6000 r.p.m. for
10 min. The fatty acid methyl esters formed were
recovered in the hexane phase and analysed by
gas chromatography (GC) on a Varian 3400 gas
chromatograph equipped with a septum-equipped
programmable injector and a flame-ionisation de-
tector. The column was a SP-2380 (30 m, 0.32 mm
i.d., 0.20 mm film thickness, Supelco, Bellafonte,
PA). The injector was programmed from 50 to
200°C at 100°C/min and held at 100°C for 3 min.
The column temperature was programmed be-
tween 75 and 190°C at 10°C/min and held at
190°C for 8 min. The chromatograms obtained
were compared with the fatty acid methyl esters
from phosphatidylcholine produced in the same
way and with lauric acid methyl ester.
2.2.2. Preparation of 1-LPG and 1-LPDHA
50 mg of phosphatidylcholine were dissolved on
2 ml of diethyl ether containing 300 mM 1-mono-
lauroyl-rac-glycerol or 1-monolauroyl-dihydroxy-
acetone. Two ml of Na-acetate buffer (0.2M), pH
5.5 containing 40 mM CaCl2, were added and the
mixtures were incubated at 25°C in an orbital

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C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
1
2.2.4. H-NMR analysis
nesulfonate (ANS) and densitometrically evalu-
ated with a Camag TLC scanner 3 (Muttenz,
Switzerland) in fluorescence/reflection mode. The
slit dimensions were set at 5×0.3 mm, the excita-
tion wavelength was 376 nm and the emission
cut-off filter was 420 nm. The relative amounts of
the substrates and the products formed were cal-
culated in each chromatographic plate from their
peak areas. Substrates and products detected from
PL D reaction samples were phosphatidylcholine,
phosphatidic acid, 1-lauroyl-phosphatidylglycerol
and 1-lauroyl-phosphatidyldihydroxyacetone with
a relative Rf of 0.10, 0.25, 0.45 and 0.65, respec-
tively. Only the ether soluble substrates and prod-
ucts were analysed for the PL C reactions and
these were 1-lauroyl-phosphatidylglycerol, 1-lau-
royl-phosphatidyldihydroxyacetone, 1-monolau-
royl-rac-glycerol, 1-monolauroyl-dihydroxyaceto-
ne and diacylglycerol with a relative Rf of
0.10, 0.15, 0.45, 0.60 and 0.70, respectively. Cali-
bration curves for all purified substrates and
products showed the detector response was linear
in the concentration range used. Relative response
factors were calculated for the quantification of
the lipids.
Structural verification of 1-LPG and 1-LPDHA
was also done by H-NMR analysis on a DRX
500c spectrometer.
1
2.2.5. Phospholipase C catalysed reactions
Typically, 1-lauroyl-phosphatidylglycerol or 1-
lauroyl-phosphatidiyldihydroxyacetone in concen-
trations between 1 and 10 mM, were dissolved in
0.2 ml diethyl ether. To the solution, 0.2 ml
HEPES buffer, pH 7.5 containing 50 mM CaCl2,
were added, with the subsequent formation of a
two-phase system. The reaction media were incu-
bated in an orbital shaker (100 r.p.m.) at 25°C.
The reactions were started by the addition of 1
unit PL C. Samples of 5 ml were withdrawn from
the ether phase at intervals and the solvent was let
to evaporate before the addition of 25 ml tetrahy-
drofuran. For the final relative quantification of
the products the remaining media was mixed with
50 ml of
1 N HCl and 500 ml of chlo-
rofrom:methanol (2:1). The mixtures were thor-
oughly vortexed and centrifuged at 6000 r.p.m.
for 10 min. The lower chloroform rich part was
collected and evaporated to a final volume of
around 30 ml. No phospholipids were detected in
the upper methanolic phase which was dis-
charged. Obtained samples were then analysed by
HPTLC.
For the final relative quantification of the prod-
ucts after extraction of the reactions with PL C,
1-lauroyl-rac-glycerophosphate, 1-lauroyl-dihy-
droxyacetonephosphate and phosphatidic acid,
the plates were eluted with the organic phase of a
2.2.6. HPTLC analysis
two-phase
mixture
of
ethyl
ac-
Thin-layer chromatography was performed on
20×10 cm silica gel 60 HPTLC plates (Merk,
Darmstadt, Germany). Then 2–15 ml samples
were applied as 6 mm bands using an autosampler
(Camag ATS3, Muttenz, Switzerland). The plates
were eluted in an unsaturated automatic develop-
ment chamber (Camag ADC, Muttenz, Switzer-
land) with the upper organic phase of a two-phase
etate:isooctane:acetic acid:water (13:2:5:10). The
calculated Rf were 0.15, 0.20 and 0.40, respec-
tively. The Rf for 1-lauroyl-rac-glycerophosphate
and 1-lauroyl-dihydroxyacetonephosphate were
considered to be the same.
3. Results and Discussion
mixture
of
ethyl
acetate:isooctane:acetic
acid:water (13:2:2:10) (Van Blitterswijk and Hilk-
mann, 1993) for the PL D reaction samples, and
3.1. Preparation of 1-LPG and 1-LPDHA and
structural characterisation
ethyl
acetate:isooctane:acetic
acid:water
(13:13:4:10) for the PL C reaction samples. Devel-
oped and dried plates were immersed for 5 s in a
dipping tank (Camag chromatogram-immersion
device III, Muttenz, Switzerland) containing a
0.1% aqueous solution of 8-anilino-1-naphthale-
The transphosphatidylation of phosphatidyl-
choline with 300 mM of 1-monolauroyl-
rac-glycerol or 300 mM of 1-monolauroyl-dihy-
droxyacetone proved to be very selective
and
no
phosphatidic
acid
was
de-

C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
179
Chromatograms of the fatty acid methyl ester
analysis of the products showed the presence of
the characteristic fatty acids in phosphatidyl-
choline and lauric acid, which accounted for one
third of the sum of the peak areas indicative for
one lauric acid in each phospholipid molecule.
After hydrolysis with PL C, the bands with an Rf
corresponding to diglyceride and lysophosphatidic
acid showed the presence of the characteristic
fatty acids from PC and methyl laurate,
respectively.
tected. The simple purification method, in which
1-LPG and 1-LPDHA precipitated in acetone,
enabled the preparation of highly pure products
and the reuse of the unreacted 1-MLG and 1-
MLDHA.
Structural verification was done by 1H-NMR
and fatty acid analysis. 1H-NMR of 1-LPG and
1-LPDHA in CDCl3 gave the following structural
information:
“ 1-Lauroyl-phosphatidylglycerol,0.88 (9H, m, 3
CH3 of fatty acids); 1.28 (60H, m, CH2 of fatty
acids); 1.6 (6H, m, 3 CH2 of fatty acids); 2.3
(6H, m; 3 COCH2 of fatty acids); 3.9–4.2 (6H,
2H, m, 4 CH2 of both glycerol molecules); 4.4
(1H, m, CH glycerol of 1-LG); 5.22 (1H, m,
CH glycerol).
“ 1-Lauroyl-phosphatidyldihydroxyacetone, 0.88
(9H, m, 3 CH3 of fatty acids); 1.28 (60H, m,
CH2 of fatty acids); 1.6 (6H, m, 3 CH2 of fatty
acids); 2.25–2.5 (4H, 2H, m; COCH2 of fatty
acids); 3.9–5.0 ( 2H, 1H, 1H, 1H, 2H, m, 4
CH2 and 1 CH of glycerol and 1-LDHA); 5.22
(1H, m, CH glycerol).
3.2. Reaction progress
Scheme 1 shows the reaction pathways for the
formation of the lysophospholipids by the combi-
nation of the enzymes phospholipase D and C.
The conditions in which the reactions are carried
out are very similar, a diethyl ether-aqueous two-
phase system for both enzymes. Phospholipase D
from Streptomyces sp. is very selective for the
formation of this transphosphatidylation product
in the presence of an alcohol. It should be men-
tioned that neither PL D from savoy cabbage nor
Scheme 1. Reaction pathways for the enzymatic two-step preparation of 1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and
1-lauroyl-dihydroxyacetonephosphate. R1: fatty acids, R2: choline, R3: lauric acid.

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C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
lower for the ketone substrate and under the same
conditions more hydrolytic product is formed.
The products of the transphosphatidylation
were purified and then treated with phospholipase
C from B. cereus. Fig. 2 illustrates the consump-
tion of 1-LPG and 1-LPDHA and the formation
of diacylglycerol. Both substrates are readily hy-
drolysed by the enzyme at similar rates. It should
be mentioned that mainly diglyceride was detected
indicating a high selectivity of the enzyme towards
the phosphoester bond between the diglyceride
and the phosphate moieties.
3.3. Kinetics of the transphosphatidylation
reaction with PL D
The transphosphatidylation of phosphatidyl-
choline with 1-monolauroyl-rac-glycerol or 1-
monolauroyl-dihydroxyacetone
followed
the
Michaelis–Menten equation profile as seen in Fig.
3. The activity of the enzyme decreased at concen-
trations higher than 75 mM 1-monolauroyl-rac-
glycerol, probably a result of substrate inhibition.
With 1-monolauroyl-dihydroxyacetone the reac-
tion rates increased in the range of concentrations
tested (5–200 mM).
Fig. 1. (a) PL D catalysed transphosphatidylation of PC with
1-monolauroyl-rac-glycerol in a two-phase system. Reaction
media containing 10 mM PC and 75 mM 1-MLG in 0.4 ml
diethyl ether, 0.4 ml Na-acetate buffer (0.2 M), pH 5.5, 40 mM
CaCl2 and 0.1 units PL D. Reaction temperature 25°C (
)
PC, (ꢀ) 1-LPG and (ꢁ) PA; (b) PL D catalysed transphos-
phatidylation of PC with 1-monolauroyl-dihydroxyacetone in
a two-phase system. Reaction media containing 10 mM PC
and 75 mM 1-MLDHA in 0.4 ml diethyl ether, 0.4 ml Na-ac-
etate buffer (0.2 M), pH 5.5, 40 mM CaCl2 and 0.1 units PL
D. Reaction temperature 25°C. ( ) PC, (ꢀ) 1-LPDHA and
(ꢁ) PA.
PL D from Streptomyces chromofoscus were effec-
tive catalysts for this transphosphatidylation
reaction.
As seen in Fig. 1a and b, the formation of
1-LPG and 1-LPDHA was predominant over the
hydrolysis of the phosphatidylcholine when 75
mM 1-MLG or 1-MLDHA was used as nucle-
ophile. Nevertheless, the selectivity seems to be
Fig. 2. Diacylglycerol formation (open symbols) in the hydrol-
ysis of 4 mM 1-lauroyl-phosphatidylglycerol and 4 mM 1-lau-
royl-phosphatidyldihydroxyacetone (closed symbols) in
diethylether-0.1 M HEPES buffer, pH 7.5 and 50 mM CaCl2
two-phase system catalysed by phospholipase C (1 U) from
Bacillus cereus (ꢂ, ꢀ) 1-LPG and (ꢃ, ) 1-LPDHA.

C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
181
1-MLG is better substrate than 1-MLDHA. The
low Km of specially 1-MLG, but also of 1-
MLDHA is remarkable. Km of nucleophiles
which are not amphiphilic, but present almost
exclusively in the aqueous phase, are usually
much higher. A value of 1.3 M has been reported
for 3-dimethylamino-1-propanol (Carrea et al.,
1997). The low Km values of the nucleophiles
used in this study may indicate that the PL D is
present either in the mixed micelles formed by the
substrate or closely associated with those micelles.
The local concentration of the nucleophile in the
vicinity of the enzyme would thus be high so that
it can react effectively. The high reactivity of the
1-MLG with respect to 1-MLDHA may be ex-
plained considering their ability of solvation and
association with the phosphatidylcholine sub-
strate. While 1-MLG is more soluble than 1-
MLDHA in the water saturated diethyl ether and
will probably easily form micelles with the phos-
pholipid, 1-MLDHA is a somewhat more rigid
molecule and with less possibilities of hydrogen
bonding, and thus less association ability.
Fig. 3. Substrate effect on the initial rates for the PL D
catalysed transphosphatidylation of PC (10 mM) with differ-
ent concentrations of 1-monolauroyl-rac-glycerol (ꢀ) and 1-
monolauroyl-dihydroxyacetone (ꢂ) in
a
diethyl ether-
Na-acetate buffer (0.2M), pH 5.5, 40 mM CaCl2 and 0.1 units
PL D. Reaction temperature 25°C.
Fig. 4 shows the final composition of the prod-
ucts at different 1-MLG and 1-MLDHA concen-
trations. The enzyme is very selective for 1-MLG,
so that at almost all substrate concentrations
tested the formation of the transphosphatidyla-
tion product was favoured. It is also noted that
PL D is not steroespecific in the synthesis of
1-LPG with respect to 1-MLG, since the nucle-
ophile, which is a racemic mixture, is almost
totally consumed at equimolar substrate
concentrations.
The selectivity of the enzyme towards the other
substrate is lower but still high considering that at
200 mM 1-MLDHA, only 8% of the product was
phosphatidic acid. Higher concentrations of wa-
ter-soluble alcohols such as glycerol or dihydroxy-
acetone are reported to be necessary for the
minimisation of phosphatidic acid formation
(Takami and Suzuki, 1994; D’Arrigo et al., 1995).
Fig. 4. Nucleophile concentration effect on the molar ratio of
the transphosphatidylation and hydrolysis products. Reaction
conditions containing 10 mM PC and different concentrations
of 1-MLG (ꢂ) or 1-MLDHA (ꢀ) on 0.4 ml diethylether, 0.4
ml Na-acetate buffer (0.2M), pH 5.5, 40 mM CaCl2 and 0.1
units PL D. Reaction temperature 25°C.
The kinetic parameters were determined by a
non-linear regression using Kaliedagraph™, Abel-
beck Software. Km (app) and Vmax (app) of 1-
MLG were 2397 and 0.9090.01 mM/min,
respectively and Km (app) and Vmax (app) for
1-MLDHA were 10495 and 0.5590.04 mM/
min, respectively. From this data it is clear that
3.4. Hydrolysis of 1-LPG and 1-LPDHA by PL
C
Fig. 5a and b illustrate the time course for the
hydrolysis of both substrates in the presence of

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C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
PL C. The calculation of the activities from the
formation of diglyceride was not accurate, since
the curves deviated greatly from linearity and thus
the determination of the kinetic parameters was
not possible. Nevertheless, the substrates were
100% hydrolysed at all concentrations tested, al-
though a sign of possible substrate inhibition was
detected with the highest concentrations. 1-
LPDHA seems to be hydrolysed more effectively
Fig. 6. Effect of phosphatidic acid on the hydrolysis of 1-lau-
royl-phophatidylglycerol by PL C form Bacillus cereus. Reac-
tion media containing 1-LPG (4 mM) and PA (") 0 mM, (ꢂ)
1mM, (ꢁ) 2 mM, (ꢃ) 3 mM and ( ) 4 mM, in 0.2 ml
diethylether, 0.2 ml HEPES buffer (0.1 M), pH 7.5, 50 mM
CaCl2 and 1 unit PL C. Reaction temperature 25°C.
than 1-LPG at low substrate concentrations. The
final relative product quantification showed
clearly that 1-lauroyl-rac-glycerophosphate and 1-
lauroyl-dihydroxyacetonephosphate were formed
as main products and that the amount of phos-
phatidic acid detected was somewhat higher (4–
5%) with 1-LPDHA than with 1-LPG (1–2%),
suggesting that the hydrolysis of the former sub-
strate is less selective.
3.5. Effect of phosphatidic acid
Phosphatidic acid was reported to completely
inhibit the hydrolysis of phosphatidylcholine and
phosphatidyldihydroxyacetone with PL C (D’Ar-
rigo et al., 1995). Since the phosphatidic acid is a
possible product in the transphosphatidylation of
PC with 1-MLG and 1-MLDHA, it was consid-
ered of interest to investigate its effect on the
hydrolysis of 1-LPG and 1-LPDHA. Fig. 6 shows
the time course for the hydrolysis of these sub-
strates in the presence of increasing amounts of
PA. As above, the determination of activities was
not possible, but clearly the lipids are hydrolysed
by the enzyme at all PA concentrations tested.
However, there is a retardation of the reaction
which could be a result of an interfacial dilution
Fig. 5. (a) Diacylglycerol formation in the hydrolysis of 1-lau-
royl-phosphatidylglycerol by PL C form Bacillus cereus. Reac-
tion media containing 1-LPG (")1 mM, (ꢂ) 2 mM, (ꢀ)
4mM, ( ) 8 mM and (ꢃ) 10 mM, in 0.2 ml diethylether, 0.2
ml HEPES buffer (0.1 M), pH 7.5, 50 mM CaCl2 and 1 unit
PL C. Reaction temperature 25°C; (b) Diacylglycerol forma-
tion in the hydrolysis of 1-lauroyl-phosphatidyldihydroxyace-
tone by PL C form Bacillus cereus. Reaction media containing
1-LPDHA (") 1 mM, (ꢂ) 2 mM, (ꢀ) 4mM, ( ) 8 mM and
(ꢃ) 10 mM, in 0.2 ml diethylether, 0.2 ml HEPES buffer (0.1
M), pH 7.5, 50 mM CaCl2 and 1 unit PL C. Reaction
temperature 25°C.

C. Virto, P. Adlercreutz / Chemistry and Physics of Lipids 104 (2000) 175–184
183
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effect (Dennis, 1983) as both substrates and PA
are surface-active substances.
Dennis, E.A., 1983. Phospholipases. In: Boyer, P.D. (Ed.), The
Enzymes, vol. XVI. Academic Press Inc, New York, pp.
323–334.
4. Conclusions
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The combination of phospholipase D and C for
the preparation of lysophosphatidic acid and 1-
lauroyl-dihydroxyacetonephosphate is simple and
effective. PL D has proven to be very selective for
the organic amphiphilic nuclophiles and conver-
sions of 100% can be achieved at low substrate
alcohol concentrations. The easy purification of
the transphosphatidylation product and the possi-
bility of reutilization of the unreacted substrates
make the system very attractive. The enzymatic
synthesis of 1-LPG and 1-LPDHA has not been
reported before and it could be of interest to
investigate their properties and possible applica-
tions. Both substances are hydrolysed by PL C in
a very selective way. The enzymatic reaction was
not affected by the presence of phosphatidic acid,
rendering unnecessary the previous purification of
the substrates. It should be the case that it is
possible to simplify the two-step reaction by the
exchange only of the aqueous phase and the en-
zyme catalyst after the completion of the first
reaction.
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phospholipase
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on lysolecithin: the structure of
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Acknowledgements
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This project was supported by the Swedish
National Board for Technical and Industrial De-
velopment (NUTEK) and the Swedish Research
Council for Engineering Sciences (TFR).
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