Natural Estolides Produced by Pseudomonas sp. 42A2 Grown on Oleic Acid: Production and Characterization

Article abstract of DOI:10.1007/s11746-003-0786-x

Estolides are a group of FA polyesters resulting from ester bond formation between a hydroxyi or olefinic group of one FA and the terminal carboxyl group of a second FA. These products are commonly found in trace amounts, forming tetraglycerides in several oil seed plants, and have been produced by acid clay and enzymatic catalysis in vitro. In this study, natural estolides produced by a bacterial culture are presented for the first time. Pseudomonas sp. 42A2 produced (E)-10-hydroxy-8-octadecenoic acid and (E)-7,10-dihydroxy-8-octadecenoic acid when grown on oleic acid. It is suggested that these FA were polymerized in culture by a lipase produced by the bacterial strain, resulting in a mixture of estolides. These compounds amounted to 3.8 g/L after 72 h of incubation. LC-MS analysis indicated that the types of estolides formed were dimers (m/z 560-610), trimers (m/z 845-906), tetramers (m/z 1122-1202), pentamers (m/z 1328-1424), and hexarners (m/z 1554-1788), with a relative abundance of 27.5, 19.4, 15, 9.7, and 11%, respectively. This is the first report in which hexamers were detected in a bacterial culture.

Full text of DOI:10.1007/s11746-003-0786-x

Natural Estolides Produced by Pseudomonas sp. 42A2
Grown on Oleic Acid: Production and Characterization
M. Peláeza, C. Orellanaa, A. Marquésa, M. Busquetsb, A. Guerreroc, and A. Manresaa,*
aLaboratori de Microbiologia, Facultat de Farmàcia and bDepartament de Bioquímica i Biologia Molecular, Facultat de
Química, Universitat de Barcelona, Barcelona, E-08028, and cDepartament Química Orgànica Biològica, Institut
d’Investigacions Químiques i Ambientals, Consejo Superior de Investigaciónes Científicas, Barcelona, E-08028, Spain
ABSTRACT: Estolides are a group of FA polyesters resulting Pseudomonas, and Alcaligenes were found suitable for es-
from ester bond formation between a hydroxyl or olefinic group
tolide synthesis in vitro using δ-lactones or hydroxy FA of
of one FA and the terminal carboxyl group of a second FA.
These products are commonly found in trace amounts, forming
tetraglycerides in several oil seed plants, and have been pro-
duced by acid clay and enzymatic catalysis in vitro. In this
study, natural estolides produced by a bacterial culture are pre-
sented for the first time. Pseudomonas sp. 42A2 produced (E)-
10-hydroxy-8-octadecenoic acid and (E)-7,10-dihydroxy-8-oc-
tadecenoic acid when grown on oleic acid. It is suggested that
these FA were polymerized in culture by a lipase produced by
the bacterial strain, resulting in a mixture of estolides. These
various chain lengths. The largest monomers described as es-
tolide components have been ricinoleic acid and 12-hydroxy-
stearic acid (7,8).
Asadauskas et al. (9) claimed great potential for specific
applications of estolides owing to their high viscosity indices
and biodegradability; they may replace certain components
of wax polyesters, lubricants, coating agents, inks, cosmetics,
and surfactants.
Here we report detecting and characterizing new bacterial
compounds amounted to 3.8 g/L after 72 h of incubation. estolides, isolated from a submerged culture of Pseudomonas
LC–MS analysis indicated that the types of estolides formed
were dimers (m/z 560–610), trimers (m/z 845–906), tetramers
(m/z 1122–1202), pentamers (m/z 1328–1424), and hexamers
(m/z 1554–1788), with a relative abundance of 27.5, 19.4, 15,
9.7, and 11%, respectively. This is the first report in which hexa-
mers were detected in a bacterial culture.
sp. 42A2 grown on oleic acid, the monomers being the hydroxy
FA derivatives (E)-10-hydroxy-8-octadecenoic acid (MHOD)
and (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD) formed
in the same culture (10).
Paper no. J10312 in JAOCS 80, 859–866 (September 2003).
MATERIALS AND METHODS
Chemicals. Technical-grade oleic acid was a gift from Lasem
(Terrassa, Spain). All chemicals and solvents of analytical
grade were purchased from Panreac (Barcelona, Spain), SDS
(Peypin, France), or Merck (Darmstadt, Germany) and used
KEY WORDS: Biotransformation, estolides, lipases, oleic
acid, polyesters, Pseudomonas.
Estolides are a group of FA polyesters resulting from ester as received. The microbiological medium, Trypticase Soy
bond formation between a hydroxyl or olefinic group of one Agar, was supplied by Pronadisa (Barcelona, Spain).
FA and the terminal carboxyl group of a second FA (1). Es-
Organism and growth conditions. Pseudomonas 42A2
tolides are commonly found in trace amounts as tetraglyc- NCIMB 40045 was originally isolated in our laboratory from
erides in several oilseed plants such as the Cruciferaceae He- oil-contaminated water. The microorganisms, after being
liophilia amplexicaulis or the Brassicaceae Lesquerella and grown on Trypticase Soy Agar medium for 24 h at 30°C, were
related genera. The oil of the Cruciferaceae has a high con- kept at 4°C, subcultured fortnightly, and preserved frozen in
tent of lesquerolic acid (14-hydroxy-cis-11-eicosenoic acid) cryovials (EAS Laboratoire, Combourg, France) at −20°C.
(2), whereas that of the Brassicaceae has a high content of
Natural estolide production and purification. An overnight
densipolic acid (15-hydroxy-9,15-octadecadienoic acid), au- culture (2% vol/vol) was used to inoculate the mineral salt
ricolic acid (14-hydroxy-11,17-eicosadienoic acid), and les- medium (MSM) of the following composition (g/L): KCl:
querolic acid (3).
0.1; KH2PO4: 1; K2HPO4: 2; CaCl2: 0.01; MgSO4·7H2O: 0.5;
Estolides were first synthesized by high-pressure conden- FeSO4·7H2O: 0.012; and NaNO3: 7. As stated below, 2%
sation of oleic acid over acidic clays (4,5). Other unsaturated (vol/vol) of technical-grade oleic acid (70% oleic acid by
FA monomers containing various functionalities on the car- GC), MHOD, or DHOD was alternatively used as carbon
bon chain such as petroselenic acid (cis-6-octadecenoic acid), source. Carbon source and mineral salt solutions were auto-
linoleic acid [9(Z),12(Z)-octadecadienoic acid], and hydroxy- claved separately. Final pH was adjusted to 6.8 with 1 N
FA have been used for estolide synthesis (4–6). Recently, li- NaOH if necessary. Submerged microbial cultures were incu-
pases isolated from Candida rugosa, Rhizomucor miehei, bated at 30°C for 72 h on a reciprocal rotary shaker (150
rev/min) in 1-L baffled Erlenmeyer flasks containing 150 mL
of medium. Samples were withdrawn from the culture and
*To whom correspondence should be addressed at Laboratori de Microbi-
ologia, Facultat de Farmàcia, Universitat de Barcelona, Joan XXIII s/n,
centrifuged at 12,000 × g, 30 min at 4°C, and the cell pellet
Barcelona, E-08028, Spain. E-mail: manresa@far.ub.es
Copyright © 2003 by AOCS Press
859
JAOCS, Vol. 80, no. 9 (2003)

860
M. PELÁEZ ET AL.
was discarded. The supernatant was acidified with HCl to pH ibrated in a 5 mM Tris-HCl buffer, pH 7.5, and eluted with a
2, and the precipitated lipids were lyophilized. The linear gradient of NaCl from 0 to 1 M. Fractions containing li-
lyophilized sample was extracted with chloroform (3×) and pase activity were eluted at a NaCl concentration of about 0.6
evaporated to dryness. The combined extracts were filtered M. The specific activity was determined as 2.5 U/mg and the
under vacuum to remove the insoluble fraction and concen- purity was checked by SDS gel electrophoresis.
trated under vacuum at 40°C in a rotary evaporator to give a
In vitro oleic estolides production. A polymer from oleic
brown oily residue. To purify the extract and isolate the poly- acid was formed in vitro by incubating 85 µg of oleic acid
mer fraction, the oily residue was dissolved in chloroform and with 30 µg of the partially purified lipase, obtained from
precipitated with chilled methanol (11). The precipitate was Pseudomonas 42A2, in 10 mL of 5 mM Tris, pH 9.2, with
separated from the monomers by centrifugation at 10,000 × 0.78 mg of sodium deoxycholate and 1 mg of CaCl2. Incuba-
g, 10 min at 4°C. The remaining solvent was removed from tion was carried out for 72 h at 30°C. The mixture was acidi-
the precipitate under a nitrogen stream, and the purified es- fied to pH 2 with HCl, and the polymer was recovered from
tolides were prepared for further analysis.
the reaction medium with chloroform.
MHOD and DHOD production and recovery. Pseudomonas
Analytical methods. Microbial growth was measured by
42A2 was incubated for 23 h in MSM as described above with the protein content of the cultures following the method of
oleic acid as carbon substrate. After this period, the culture was Lowry et al. (12) against a standard of BSA (Sigma Chemi-
centrifuged at 12,000 × g, 30 min at 4°C, and the pellet was dis- cal Co., St. Louis, MO).
carded. The supernatant was acidified to pH 3 with 2 N HCl,
Lipase activity was determined in an in vitro assay that was
and subsequently lipids were extracted twice with 2 vol of chlo- based on the decrease in turbidity when a lipase hydrolyses
roform (1:2 vol/vol). The organic phases were collected and emulsified triolein. The decrease in turbidity was monitored by
dried with anhydrous Na2SO4, and the solvent was removed to following the changes of A340nm. The reaction mixture com-
dryness in a rotary evaporator. To obtain MHOD and DHOD, prised: 26 mmol/L Tris buffer, pH 9.2; 19 mmol/L sodium des-
the crude lipid extract was purified by column chromatography oxycholate; 0.1 mmol/L calcium chloride; and 0.30 mmol/L
on a 50 × 3 cm i.d. column packed with 60 g of silica gel G 60A triolein. The standard reaction mixture also contained 3.0 mg/L
C.C. (SDS, Peypin, France). The column (retention volume = colipase. The assay was carried out at 37°C. Lipase activity
140 mL) was successively eluted with 140 mL of the follow- (U/L) of a sample was calculated by comparing the rate of
ing hexane/diethyl ether (vol/vol) mixtures: 100:15, 100:25, change of A340nm of the sample to that of a standard lipase sam-
100:35, 100:45, 100:60, 100:75. Two retention volumes (i.e., ple of known activity, as described in the manual of the LIP
280 mL) of a 100:100 (vol/vol) hexane/diethyl ether solution MPR 1 (Cat. No. 159697; Boerhinger-Mannheim, Mannheim,
were then used to conclude the gradient elution. All hexane di- Germany). Residual nitrate content of the medium was mea-
ethyl ether solutions were acidified by adding 1 mL of acetic sured using a specific nitrate test (Aquamerck 8032; Merck).
acid per 140 mL mixture. Finally, the column was eluted with
Purified estolides were subjected to hydrolysis with 0.5 N
methanol. A total of 40 fractions was collected and analyzed NaOH at room temperature for 24 h. Before GC analysis,
by TLC. TLC analyses were performed on 20 × 20 cm silica samples were methylated with diazomethane (13) and diluted
gel 60 (0.25 mm thickness) analytical plates (Panreac, Spain). with ether.
Plates were developed with 80:50:1 chloroform/methanol/
Methyl esters of the monomers were analyzed by GC
acetic acid (by vol), and spots were visualized by spraying analysis on a Shimadzu GC-14A gas chromatograph (Shi-
with a solution of phosphomolybdic acid in absolute ethanol, madzu, Kyoto, Japan) equipped with a split-splitless injector
followed by charring with a heat gun. Those fractions con- and a FID. A Supelco Omegawax™ 250, 30 m × 0.25 mm ×
taining compounds corresponding to the Rf similar to the 0.25 µm capillary column was used (Supelco, Bellefonte,
MHOD and those corresponding to the Rf of the DHOD PA). The oven temperature was kept at 210°C for 3 min, fol-
were combined and analyzed in a Shimadzu LC 9A gradient lowed by an increase of 10°C/min to 240°C, held for 8 min,
system (Shimadzu, Kyoto, Japan). Pure MHOD was found in before initiating a second program of 2°C/min to 248°C, and
fractions eluted with mixtures of 50:50 hexane/diethyl ether, then held for 20 min. Peaks were recorded on a Shimadzu
whereas pure DHOD eluted in methanol fractions.
C-R4 Chromatopac Integrator. Analyses were carried out on
Lipase production and recovery. Pseudomonas 42A2 was the methyl esters of the organic extract of the cell-free cul-
incubated in MSM as described above with oleic acid as car- ture. Methyl ricinoleate (Sigma) of known concentration was
bon substrate for 72 h. After centrifugation at 12,000 × g for used as standard. Retention times of MHOD and DHOD were
30 min at 4°C the cells were discarded. The enzyme was puri- 11.2 min and 25.3 min, respectively.
fied from the culture media by 80% ammonium sulfate precip-
IR spectra were recorded on a Nicolet model 510 with a
itation with constant stirring for 10 min, and the mixture was Fourier transform spectrometer (Madison WI).
then centrifuged at 17,000 × g for 15 min at 4°C. Immediately
1H and 13C NMR spectra were obtained in CDCl3 solutions
after, the sample was resuspended in 5 mM Tris-HCl, pH 7.5, on a Varian spectrometer, operating at 250 and 300 MHz for
and subsequently dialyzed against the same buffer for 24 h at 1H and 62.5 and 75 MHz for 13C, and the values are expressed
4°C. The sample was applied to a column of Q-Sepharose Fast in δ scale relative to internal tetramethylsilane. For the 250
Flow (Amersham, Cerdanyola, Spain) (25 × 2.6 cm) pre-equil- MHz spectrometer the following conditions were used:
JAOCS, Vol. 80, no. 9 (2003)

PRODUCTION OF ESTOLIDES BY PSEUDOMONAS SP. 42A2
861
temperature 27°C, acquisition time 6,396 s, pulse 39.37, and
spectral width 5123 Hz. In the case of the 300 MHz spectra,
the conditions were: temperature 27°C, pulse 39.37 for 1H and
43.27 for 13C, acquisition time 1,998 s, spectral width 4500.5.
EI-MS were run on the trimethylsilyl ethers of the methyl
ester derivatives (14) using a Hewlett-Packard mass spectrom-
eter coupled to a gas chromatograph. The GC was equipped
with a HP-5MS, 30 m × 0.25 mm × 0.25 mm capillary column
(Hewlett-Packard, Palo Alto, CA). The oven temperature was
kept at 140°C for 3 min, followed by a program of 5°C/min to
210°C, held for 8 min, and a second program of 10°C/min to
240°C, kept for 8 min; a final program of 2°C/min raised the
oven temperature to 248°C, which was maintained for 10 min.
HPLC was performed on a Shimadzu LC9A gradient-capa-
ble system equipped with a Hypersil WP-300 C8 150 × 0.46
mm reversed-phase column (Teknokroma, S. Cugat, Spain).
The solvent gradient was programmed from a 1:1 mixture of
acetonitrile/water containing 0.1% acetic acid to pure acetoni-
trile at a flow rate of 1 mL/min. The chromatograph was cou-
pled to an ELSD (Sedere, Alfortville, France) set at 50°C and
2 bar. Standards used were oleic acid, MHOD, and DHOD.
In the HPLC–electrospray (ES–MS) system, postcolumn ad-
dition of acetone at 200 µL/min was carried out using a syringe
pump. Mobile phase and acetone were mixed in a tee valve, and
a split system (1:50) was used to introduce the effluent into the
ES. MS was performed using a quadrupole mass spectrometer
equipped with a pneumatically assisted electrospray (ES) ion
source. The positive ion mode was used. Full scan data were ob-
tained by scanning from m/z 100 to 2900 in compressed cen-
troid mode using a cycle time of 3.3 s and an interscan time of
0.34 s. The working conditions for the ES were as follows: dry
nitrogen was heated to 100°C and introduced into the capillary
region at a flow rate of 400 L/h. The capillary was held at a po-
tential of 3.5 kV and the extraction voltage was −30 V.
FIG. 1. Time course of submerged culture of Pseudomonas sp. 42A2 in
mineral medium with 2% oleic acid as carbon source incubated at 30°C
for 70 h. (A) cell protein (●), (E)-10-hydroxy-8-octadecenoic acid
(MHOD) (▲), (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD) (✱),
oleic acid (■). (B) extracellular lipase (●), estolides (▲), cell protein (●),
residual NaNO3 (◆).
RESULTS AND DISCUSSION
Time course of estolide production. As reported earlier, when been found in the literature, these data suggested that the se-
Pseudomonas 42A2 was cultivated in MSM with oleic acid creted lipase might potentially catalyze polymerization of the
as the sole carbon source, high amounts of the hydroxy FA oleic acid and the hydroxy FA accumulated in the supernatant
derivatives MHOD and DHOD were produced in the extra- with the consequent formation of new natural estolides. On
cellular medium (10,15). The time course of the process is these premises, we studied the presence of estolides in the cul-
shown in Figure 1. After 24 h of incubation, oleic acid (Fig. ture medium. Lipase activity was detected early in the culture,
1A) and the nitrogen source (Fig. 1B) were depleted from the increasing sharply to 124 U/L at the end of the experiment (Fig.
culture medium; thereafter, no significant bacterial growth 1B). Estolides were detected after 5 h of incubation, and the
was observed. Total cell protein reached 1.8 g/L. Analysis of amount of polymer increased with the lipase content in the cul-
the culture supernatant products showed that the main hy- ture, reaching 3.8 g/L at the end of the culture. They were iso-
droxy FA formed were MHOD (maximum yield: 5.8 g/L at lated by organic extraction from the culture medium, yielding
25 h) with a cellular yield of YMHOD/cell protein = 3.2 (w/w) and a white powder. This hypothesis of lipase-catalyzed estolide
a maximum of 2.6 g/L of DHOD at 23 h of incubation, with a formation was reinforced by the fact that estolides from oleic
cellular yield of YDHOD/cell protein = 1.4 (w/w). Nevertheless, acid were obtained when purified lipase from Pseudomonas
after 50 h of incubation, neither MHOD nor DHOD was de- 42A2 culture supernatant was used, as stated above.
tected in the culture supernatant.
Characterization of natural estolides. At the end of the
As previously reported, an extracellular lipase was detected culture (72 h) of Pseudomonas 42A2 on technical-grade oleic
in the cultures of this strain when incubated in mineral medium acid, cells were collected by centrifugation and removed.
with frying oils as carbon source (16). Although no report has After acidification, solvent extraction of the supernatant and
JAOCS, Vol. 80, no. 9 (2003)

862
M. PELÁEZ ET AL.
FIG. 2. 1H (A) and (B) 13C NMR (300 MHz) spectra of estolides produced by Pseudomonas sp. 42A2 when grown
in mineral media with oleic acid as carbon source.
subsequent methanol precipitation furnished the natural es- mers, i.e., O = oleic acid, M = MHOD, and D = DHOD. As
tolide, whose purity was determined by HPLC (see the Mate- shown in the chromatogram, estolides with higher M.W.
rial and Methods section). The IR spectrum revealed the pres- eluted at longer retention times. The first section of the chro-
ence of ester and acid groups as well as a trans unsaturation. matogram (elution time 5.6 to 12.0 min) corresponded to
IR bands (cm−1) observed were: 3648, 3432, 2946, 1741, dimers (E1) of M.W. between m/z 560 (i.e., O-D) and 610 (i.e,
1712, 1231, and 970 (trans alkene) cm−1. The polymeric na- D-D). The second part of the chromatogram (elution time
ture of the estolide was confirmed by its NMR spectra pre-
sented in Figures 2A and 2B. 1H NMR peaks (δ) were: 5.62
(m, CHOH–CH=CH–CHOH), 4.90 (b, CHOCOR), 4.05 (m,
CHOH–CH=CH), 2.32 (t, J = 7.2 Hz, CH2CO2R). 13C NMR
(ppm): δ 178.0 (CO2H), 169.0 (CO2R), 72.4 (CHOH), 70.8
(CHOCO). A fraction of the purified estolides was subjected
to base hydrolysis (0.5 M KOH/CH3OH), and the resulting
monomers were silylated and methylated. GC–MS analysis
confirmed the presence of the expected monomers in the fol-
lowing ratio: oleic acid, 10–20%; MHOD, 44–56%; and
DHOD, 17–24%.
Owing to the presence of the three monomers in the cul-
ture medium (oleic acid, MHOD, and DHOD), several classes
of estolides can be formed, and thus a wide range of polymers
with various M.W. were found in each type of estolide. As an
example, in Scheme 1 are represented the possible types of
estolides that can be found in the culture supernatant for
Pseudomonas 42A2 grown in mineral medium with 2% oleic
acid as carbon source at 30°C for 75 h. The purified organic
extract from Pseudomonas 42A2 culture after 72 h of incuba-
tion was analyzed by HPLC–MS (Fig. 3). Peak assignments
were based on M.W. in the MS spectra considering the possi-
ble combinations of the M.W. of the corresponding mono-
SCHEME 1
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PRODUCTION OF ESTOLIDES BY PSEUDOMONAS SP. 42A2
863
FIG. 2. (continued).
14.0–16 min) corresponded to trimers (E2) of M.W. between
m/z 845 and 906 (i.e., M-D-D; O-M-M; O-O-D; or O-D-D).
In the third section of the chromatogram, tetramers (E3) of
molecular mass between m/z 1122 and 1202 (i.e., O-O-D-D;
O-D-D-D; M-M-D-D; or D-D-D-D) were eluted between
16.2 and 18.6 min. Between 18.8 and 21.8 min, the fourth part
of the chromatogram, pentamers (E4), eluted with a range of
M.W. between m/z 1328 and 1498 (i.e., O-O-O-O-O; O-O-D-
D-D; M-M-D-D-M; M-M-M-M-M). In the last section of the
chromatogram (elution time 22.0 to 23.2 min) appeared the
largest estolide (E5) hexamer-type (Fig. 4) with an apparent
M.W. of approximately m/z 1554 to 1788 (i.e., O-M-M-O-M-
M or O-D-D-O-D-D; M-M-D-M-M-D; M-D-D-M-D-D). The
calculated relative abundance of estolides was: dimers 27.5%,
trimers 19.4%, tetramers 15.0%, pentamers 9.7%, and hexa-
mers 11.0%. It should be noted that an average of tetramers
has been obtained by enzymatic synthesis (5,10), whereas the
largest analytes detected in Pseudomonas 42A2 culture were
hexamers. To our knowledge, no other reports to date have
described natural polymers composed of six monomers.
The polymerization of MHOD was carried out by incu-
bating Pseudomonas 42A2 for 72 h using MHOD as carbon
substrate in MSM. After acidic precipitation (HCl, pH 2)
of the culture supernatant and subsequent lyophilization,
the residual white powder was extracted with chloroform
and purified by methanol precipitation. The purified polymer
was analyzed by IR and the following bands were observed:
3412, 2926, 1710, 1242, 1184, 1096, 969 (trans alkene),
and 724 cm−1. 1H NMR (CDCl3) peaks (δ) were as follows:
FIG. 3. LC-MS chromatogram of the purified organic extract from the
supernatant of Pseudomonas sp. 42A2 grown in mineral salt medium
with oleic acid at 30°C for 75 h. E1: section of dimers; E2: section of
trimers; E3: section of tetramers; E4: section of pentamers; E5: section of
hexamers.
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864
M. PELÁEZ ET AL.
m/z
FIG. 4. MS of the estolides fraction E5 obtained from Pseudomonas sp. 42A2 culture grown in mineral medium with technical-grade oleic acid as
carbon source. For abbreviation see Figure 3.
(CHOCO). The poly-DHOD was similarly hydrolyzed (0.5 M
KOH/CH3OH ), rendering back the expected monomer, which
was characterized by its H NMR spectrum. GC–MS of the
5.8–5.0 (complex absorption with two multiplets at δ 5.35 and
5.67 ppm corresponding to CHOHCHa=CHb surpassed), 2.35
1
(t, CH2CO2R), 2.04 (m, CH2C=C) (Fig. 5). The following 13
C
silylated methyl ester of the monomer was also identical [main
fragments at m/z (%) 359 (11), 343 (10), 269 (15), 253 (14),
231(7), and 215 (7)] to that described elsewhere (10), confirm-
ing the structure of the DHOD monomer.
In an attempt to understand or explain the behavior of the
lipase in the bulk of the culture, we suggest that mixed mi-
celles or supramolecular aggregates are formed in the culture
due to the presence of oleic acid, MHOD, and DHOD (which
have surfactant properties) in which the lipase may be trapped
within the hydrophobic domain of such aggregates.
NMR signals (δ) were observed: (CDCl3): 179.1 (COOH),
173.2 (COOR), 125.4, 136.0–128.0 (olefinic C clusters), and
70.4 ppm. Basic hydrolysis (0.5 M KOH/EtOH) of the
polymer followed by methylation and silylation gave two
major peaks, one of which was identified as methyl
10-trimethylsilyloxy-8-octadecenoate by the characteris-
tically intense fragments at m/z (%) 271 (64) and 241
(17), corresponding to [Me3SiOCHCH=CH(CH2)6CO2CH3]+
and [HC=CHCH(OSiMe3)(CH2)7CH3]+, respectively. A frag-
ment of m/z 129 (27) may be assigned to [(CH2)5CO2CH3]+
from allylic cleavage of the molecular ion, and/or to
[CH2=CHCHOSiMe3]+ from further cleavage of ions at m/z
241 or 271.
Likewise, when DHOD was used as substrate, a new poly-
mer was obtained from the culture medium. The IR spectrum
showed bands at ν 3426, 2926, 1712, 1235, 1155, 1072, and 971
(trans alkene) cm−1. The 1H NMR spectrum (Fig. 6) had the fol-
lowing main features: δ 5.62 (m, CHOH–CH=CH-CHOH),
5.18 (m, CH=CHCHOCO), 4.10 (CHOH–CH=CH–CHOH),
and 2.34 (t, CH2–CO2R). The 13C NMR showed bands at
δ 178.6 (CO2H), 170.0 (CO2R), 72.3 (CHOH), and 70.9
Enzymatic preparation of estolides. Oleic acid estolide
was obtained by the enzymatic action of the lipase obtained
from Pseudomonas 42A2 culture on pure oleic acid. After pu-
rification and methylation, the methylated polyoleic acid
showed IR bands at 2929, 1728, 1275, 1123, and 958 cm−1.
The presence of the characteristic bands for polyoleic acid in
1
the H NMR spectrum at δ 4.8 ppm, corresponding to the
methine proton CHOCOR, at δ 2.25 of the α-carboxylic pro-
tons, the 74.0 ppm of the methine carbon in the 13C NMR,
and the two carbonyl carbons at 179.7 ppm of the acid and
173.7 ppm of the estolide, identical to the main features of the
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PRODUCTION OF ESTOLIDES BY PSEUDOMONAS SP. 42A2
865
FIG. 5. 1H NMR spectrum of estolides produced by Pseudomonas sp. 42A2 when grown in aerated culture at 30°C
in mineral medium with MHOD as carbon source. For abbreviation see Figure 1.
FIG. 6. 1H NMR spectrum of estolides produced by Pseudomonas sp. 42A2 when cultivated in aerated culture at
30°C in mineral medium with DHOD as carbon source. For abbreviation see Figure 1.
JAOCS, Vol. 80, no. 9 (2003)

866
M. PELÁEZ ET AL.
Hydroxy Acids and Estolide Triglycerides of Heliophila am-
plexicaulis L. f. Seed Oil, Lipids 14:576–579 (1978).
3. Hayes, D.G., R. Kleiman, and B.S. Phillips, The Triglyceride
Composition, Structure and Presence of Estolides in the Oil of
Lesquerella and Related Species, J. Am. Oil Chem. Soc. 72:
559–569 (1995).
polyestolide reported by Isbell et al. (18), indicated that our
crude lipase catalyzed the polymerization of oleic acid in
1
vitro. Other H NMR bands appeared at δ 5.34 (t, 2H,
CH=CH), 4.89 (t, CHOCOR), 3.66 (s, 3H, CO2CH3), 2.4–2.2
(m, CH2COOR), and 2.00 (m, CH2C=C), whereas in the 13
C
4. Isbell, T.A., R. Kleiman, and S.M. Erhan, Characterization of
Monomers Produced from Thermal High-Pressure Conversion
of Meadowfoam and Oleic Acids into Estolides, Ibid. 69:
1177–1183 (1992).
5. Erhan, S.M., R. Kleiman, and T.A. Isbell, Estolides from
Meadow Foam Oil Fatty Acids and Other Monounsaturated
Fatty Acids, Ibid. 70:641–645 (1993).
6. Fehling, E., Analysis of Estolides in Technical Hydroxylates
Fatty Acids from Plant Oils, Ibid. 72:355–359 (1995).
7. Hayes, D.G., The Catalytic Activity of Lipases Toward Hydroxy
Fatty Acids—A Review. Ibid. 73:543–549 (1996).
8. Yoshida, Y., M. Kawase, C. Yamaguchi, and T. Yamane, Enzy-
matic Synthesis of Estolides by a Bioreactor, Ibid. 74:261–267
(1997).
9. Asadauskas, S., T.P. Abbott, and T.A. Isbell, Biodegradable
Oleic Estolide Ester Base Stocks and Lubricants, U.S. Patent
6,018,063 (2000).
10. Guerrero, A., I. Casals, M. Busquets, Y. León, and A. Manresa,
Oxidation of Oleic Acid to (E)-10-Hydroperoxy-8-octadecenoic
Acid and (E)-10-Hydroxy-8-octadecenoic Acids by Pseudo-
monas sp. 42A2, Biochim. Biophys. Acta 1347:75–81 (1997).
11. Cromwick, A., T. Foglia, and R.W. Lenz, The Microbial Pro-
duction of Poly(hydroxyalkanoates) from Tallow, Appl. Micro-
biol. Biotech. 46:464–469 (1996).
12. Lowry, O.H., N. Rosebrough, A.L. Farr, and A.L. Randall, Pro-
tein Measurement with the Folin Phenol Reagent, J. Biol. Chem.
193:265–257 (1951).
13. Moore, J.A., and D.E. Reed, Diazomethane, in Organic Synthe-
sis, edited by V. Baumgarten, Vol. 5, John Wiley & Sons, New
York, 1973, pp. 351–355.
14. Sweelwy, C.C., R. Bentley, M. Makita, and W.W. Wells,
Gas–Liquid Chromatography of Trimethylsilyl Derivatives of
Sugars and Related Substances, J. Am. Oil Chem. Soc. 85:
2497–2507 (1963).
15. Culleré, J., O. Durany, M. Busquets, and A. Manresa, Biotrans-
formation of Oleic Acid into (E)-7-Hydroxy-8-octadecenoic
acid and (E)-7,10-Hydroxy-8-octadecenoic Acid in an Immobi-
lized System, Biotechnol. Lett. 23:215–219 (2001).
16. Haba, E., M.J. Espuny, M. Busquets, and A. Manresa, Screen-
ing and Production of Rhamnolipids by Pseudomonas aerugi-
nosa 47T2 NCIB 40044 from Waste Frying Oils, Enzyme Mi-
crob. Technol. 23:215–219 (2000).
17. Hayes, D.G., and R. Kleiman, Lipase-Catalyzed Synthesis and
Properties of Estolides and Their Esters, J. Am. Oil Chem. Soc.
72:1309–1316 (1995).
18. Isbell, T.A., and R. Kleiman, Characterization of Estolides Pro-
duced from the Acid-Catalyzed Condensation of Oleic Acid,
Ibid. 71:379–383 (1994).
NMR, the remaining peaks resonated at δ 174.7, 130.8, 130.0,
129.7, 128.7, 73.1, 71.9, 51.5, 48.2, 47.3, and 42.0 ppm.
From the results presented above it is obvious that estolide
formation has occurred. The route of this reaction may lie in
the autoxidation of oleic acid in the aqueous and aerated re-
action system. These oxidized derivatives of oleic acid are
then postulated to form estolides in an enzymatic reaction.
In a similar fashion, enzymatic formation of estolides from
ricinoleic acid, linoleic acid, palmitic acid, stearic acid, or di-
hydroxystearic acid has been described using free lipases
from Candida and Pseudomonas sp. in an aqueous system or
reverse micelles (8,17). On the other hand, Yoshida et al. (8),
when using immobilized lipase in an organic medium, re-
ported a strong inhibition of the enzyme activity due to the
presence of water.
With few exceptions, 1,3-positional-specific lipases can-
not form estolides because they can rarely utilize secondary
alcohols as substrate (18). However, the lipases from
Pseudomonas species are non-positional-specific and may
catalyze esterification reactions using aliphatic acids or lac-
tones in aqueous environments (19).
This is the first report of natural estolides being detected
and characterized in a bacterial culture and characterized.
Probably owing to the presence of an extracellular lipase,
when grown on oleic acid Pseudomonas 42A2, induced the
formation of several species of estolides. The monomers
found in these estolides were: oleic acid, MHOD, and DHOD.
When the carbon substrates were MHOD or DHOD, the es-
tolides produced were composed of the unique monomer. The
degree of polymerization, up to a hexamer, is the largest re-
ported so far. The precipitated lipase catalyzed the formation
of estolide in vitro.
ACKNOWLEDGMENTS
This research was supported by the Comissió Interdepartamental de
Recerca i Innovació Tecnològica (199956R 00024, 2001SGR-0342,
and 2001SGR 00143) and Comisión Interdepartamental de Ciencia
y Tecnología (PPQ-2000-0105-P4-03, REN 2001-3224, and AGL
2000-1695-C02-01) projects.
19. Dong, H., H.-d. Wang, S.-g. Cao, J.-c. Shen, Lipase-Catalyzed
Polymerization of Lactones and Linear Hydroxyesters, Biotech-
nol. Lett. 20:905–908 (1998).
REFERENCES
1. Erhan, S.M., R. Kleiman, and T.P. Abbott, Quantitation of Es-
tolides by Fourier Transform Infrared Spectroscopy, J. Am. Oil
Chem. Soc. 73:563–567 (1996).
2. Plattner, R.D., K. Payne-Wahl, L.W. Tjarks, and R. Kleiman,
[Received April 26, 2002; accepted May 15, 2003]
JAOCS, Vol. 80, no. 9 (2003)