Identification of (E)-11-hydroxy-9-octadecenoic acid and (E)-9-hydroxy-10-octadecenoic acid by biotransformation of Oleic acid by pseudomonas sp. 32T3

Article abstract of DOI:10.1007/s11746-001-0310-3

Pseudomonas sp. 32T3, a newly identified strain originally isolated from a vegetable oil-contaminated soil, produces three monohydroxy acids-(E)-11-hydroxy-9-octadecenoic acid, (E)-10-hydroxy-8-octadecenoic acid, and (E)-9-hydroxy-10-octadecenoic acid-as

Full text of DOI:10.1007/s11746-001-0310-3

Identification of (E)-11-Hydroxy-9-octadecenoic Acid
and (E)-9-Hydroxy-10-octadecenoic Acid by Biotransformation
of Oleic Acid by Pseudomonas sp. 32T3
E. Rodrígueza, M.J. Espunya, A. Manresaa,*, and A. Guerrerob
aLaboratori de Microbiologia, Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain.,
and bDepartamento Química Orgànica Biológica, IIQAB, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
ABSTRACT: Pseudomonas sp. 32T3, a newly identified strain originally isolated from a vegetable oil-contaminated soil.
originally isolated from a vegetable oil-contaminated soil, pro-
duces three monohydroxy acids—(E)-11-hydroxy-9-octade-
cenoic acid, (E)-10-hydroxy-8-octadecenoic acid, and (E)-9-hy-
droxy-10-octadecenoic acid—as bioconversion products of
oleic acid. The bacterial cells were grown in a mineral medium
containing oleic acid as the main carbon substrate. The com-
pounds were identified as the corresponding methyl esters on
the basis of their chromatographic and spectroscopic (1H and
13C nuclear magnetic resonance and gas chromatography–mass
spectrometry) features.
This strain produces the hydroxy derivatives (E)-11-hydroxy-
9-octadecenoic acid, (E)-10-hydroxy-8-octadecenoic acid,
and (E)-9-hydroxy-10-octadecenoic acid when grown in a
mineral medium with oleic acid as the main carbon substrate.
In this work we present the detection and chemical structural
determination of hydroxy fatty acids obtained from cultures
of Pseudomonas sp. 32T3 when incubated in salts mineral
medium with crude oleic acid.
Paper no. J9784 in JAOCS 78, 593–597 (June 2001).
MATERIALS AND METHODS
KEY WORDS: Biotransformation, hydroxy fatty acids, oleic
acid, Pseudomonas.
Microorganism and culture medium. Pseudomonas sp. 32T3
was isolated from a vegetable oil-contaminated soil sample.
The medium composition was as follows (g/L): K2HPO4, 2;
KH2PO4, 1; KCl, 0.1; MgSO4·7H2O, 0.5; CaCl2, 0.01;
Hydroxy fatty acids are an important family of compounds FeSO4·7H2O, 0.012; NaNO3, 7; and trace minerals, 0.05 mL.
with a wide range of applications, such as resins, surfactants, The medium was sterilized for 20 min at 121°C, and techni-
lubricant additives, plastics, coatings, cosmetics, nylons, and cal-grade oleic acid, previously sterilized in the same condi-
materials for the textile industry. Moreover, they protect tions, was added to 2% (vol/vol) as the carbon source. The
plants against microbial infection, although the mechanism pH was adjusted to 6.8.
of these antimicrobial effects is poorly understood (1).
Chemicals. All solvents and chemicals were analytically
Bioconversion of oleic acid by a variety of microorgan- pure and obtained from commercial sources (Fluka, Stein-
isms has been reported. The process may involve saturation heim, Switzerland; Panreac, Barcelona, Spain; and Aldrich,
of the alkyl chain, oxidation of the double bond to the ke- Madrid, Spain). The substrate for biotransformation, techni-
toacid by Staphylococcus (2), Nocardia, Mycobacterium (3), cal-grade oleic acid (ca. 80% purity by gas chromatography),
or Sphingobacterium (4), oxidation to hydroxystearic acid by was kindly supplied by Clarian (Barcelona, Spain).
Saccharomyces (3) or Rhodococcus (5) or to several hydrox-
Bioconversion and product extraction. The above culture
yoctadecenoic acids by Nocardia, Selenomonas, and Entero- medium was inoculated with 2% (vol/vol) of a Pseudomonas
coccus (6,7) or Alcaligenes sp. (8). Hydroxy ketoacids have sp. 32T3 saline suspension (optical density at 540 nm of 2)
also been produced by a Bacillus strain (9). We reported that from an overnight culture. The culture was aerobically grown
Pseudomonas sp. 42A2 produces (E)-10-hydroxy-8-octa- at 30ºC in 250 mL of medium in a 1-L Erlenmeyer flask
decenoic acid and (E)-7,10-dihydroxy-8-octadecenoic acid shaken at 150 rpm in a reciprocal shaker (Sanyo, Braun
and postulated that the formation of the monohydroxy deriv- Biotech, Barcelona, Spain). Cultures were incubated for 120
ative may occur through the corresponding hydroperoxide h and samples were taken at 24, 48, 72, 96, and 120 h. The
precursor (10–12). The diol derivative was also identified by organic extracts were analyzed by thin-layer chromatography
Hou et al. (13) from cultures of Pseudomonas aeruginosa, (TLC), and the amount of bioconversion products was high-
and later the absolute configuration (14) of the monohydroxy est after 72 h of incubation. At the end of the incubation, the
derivative was also determined to be S (15).
culture was centrifuged, and the cell pellet was discarded. The
In our current interest in the microbiological reutilization supernatant was acidified to pH 2 with 6 N HCl and extracted
of waste oils, we report a new strain Pseudomonas sp. 32T3, twice with chloroform/methanol (2:1 vol/vol). The combined
extracts were dried over anhydrous sodium sulfate and fil-
tered, and the solvent was removed with a rotary evaporator.
*To whom correspondence should be addressed.
E-mail: manresa@farmacia.far.ub.es
Copyright © 2001 by AOCS Press
593
JAOCS, Vol. 78, no. 6 (2001)

594
E. RODRÍGUEZ ET AL.
Purification of products. The crude lipid extract was ester- ture for 20 min more. Helium was used as the carrier gas at a
ified with diazomethane (16) and purified by column chroma- flow rate of 0.60 mL/min.
tography on a 50 × 3 cm i.d. column packed with 60 g of sil-
1H and 13C NMR spectra were obtained in CDCl3 on a
ica gel 60 A C.C. Chromagel (SDS, Peypin, France). The Varian Gemini-300 spectrometer (Varian Associates, Palo
1
column was eluted with mixtures of hexane/diethyl ether Alto, CA) operating at 300 MHz for H and 75.4 MHz for
from 100:15 to 100:100 (all ratios vol/vol), increasing the 13C. The values are expressed in δ scale relative to the chlo-
amount of ether by 10% in each retention volume. A total of roform signal (δ 7.26 ppm) of the nondeuterated material.
60 fractions was eluted and analyzed by TLC, and those frac-
tions containing compounds with Rf similar to those of the ex-
pected monohydroxy octadecenoates were combined. These
RESULTS AND DISCUSSION
corresponded to fractions eluted with mixtures of hexane/di- Identification of the microorganism. The original isolate was
ethyl ether 100:60, 100:75, and 100:90. The pooled material obtained from a sample taken from an oil-contaminated soil
was repurified by column chromatography, eluting with after an enrichment culture with kerosene and plated on Tryp-
mixtures of hexane/diethyl ether 100:5 to 100:100 to collect ticase Soy Agar (TSA, Pronadisa, Barcelona, Spain). The iso-
only the eluate with hexane/diethyl ether 100:40. This late was a Gram-negative rod with respiration metabolism. It
fraction was analyzed by gas chromatography–mass spec- was catalase, oxidase, and nitrate-reduction positive and ca-
troscopy (GC–MS) and 1H and 13C nuclear magnetic pable of growing on citrate. It was Voges-Proskauer, indole,
resonance (NMR).
and gelatin-hydrolysis negative. The isolated strain 32T3 was
Analysis of products. TLC analyses were performed on 20 motile. Based on these results, the isolate was assigned to the
× 20 cm silica gel 60 F254 (0.25 mm thickness) analytical genus Pseudomonas.
plates (Panreac) and 20 × 20 cm silica gel 60 F254 (0.50 mm
Biotransformation of oleic acid by Pseudomonas sp. 32T3.
thickness) preparative plates with a 20 × 4 cm concentration In previous studies we reported that Pseudomonas sp. 42A2
zone (Merck, Darmstadt, Germany). Plates were eluted with converted oleic acid into (E)-10-hydroxy-8-octadecenoic
hexane/diethyl ether/acetic acid (80:50:1, vol/vol/vol), and acid, (E)-10-hydroperoxy-8-octadecenoic acid, and (E)-7,10-
spots were visualized by spraying with a solution of phos- dihydroxy-8-octadecenoic acid (11). In our efforts to identify
phomolybdic acid in absolute ethanol, followed by charring new hydroxy fatty acid derivatives from oleic acid, the strain
with a heat gun. GC analyses were carried out by inject- Pseudomonas sp. 32T3 was cultivated in mineral medium
ing the samples into a Shimadzu GC-14A gas chromato- with technical-grade oleic acid as substrate for growth and
graph (Shimadzu, Kyoto, Japan) equipped with a flame-ion- biotransformation. Detection of the biotransformation prod-
ization detector and a Supelco SPB-1 fused-silica capillary ucts was carried out by comparison of their TLC behavior
column (30 m × 0.25 mm i.d., 0.25 µm film thickness, from with that of the products of biotransformation of oleic acid by
Tecknokroma, St. Cugat, Spain). The integration of peaks was Pseudomonas sp. 42A2 (11). However, since the aim of this
carried out on a Shimadzu C-R4A integrator-recorder. Helium work was the characterization of monohydroxy derivatives
was used as the carrier gas at a flow rate of 0.98 mL/min. from a new bacterial strain, we have not quantitatively esti-
Compounds were isothermally eluted at 200ºC.
mated the yield of the biotransformation products.
For identification purposes, the compounds were trans-
Identification of the conversion products. Preliminary
formed into their corresponding trimethylsilyl (TMS) deriva- structure determination was performed by GC analysis of
tives as follows. In a 25-mL Erlenmeyer flask were placed the methylated crude extract and revealed the presence, among
1–5 mg of the sample, 1 mL of anhydrous pyridine, 0.2 mL others, of two peaks with retention times of 13.723 and 14.983
of hexamethyldisilazane, and 0.1 mL of trimethylchlorosi- min on a SPB-1 fused-silica capillary column (Fig. 1A). One
lane. The reaction mixture was stirred for 12 h at room tem- peak had a retention time similar to that of the methyl ester
perature; then 5 mL of hexane was added, quenched with 5 of (E)-10-hydroxy-8-octadecenoic acid (14.3 min) (12),
mL of water, and decanted. The aqueous layer was extracted suggesting that these products were monohydroxy fatty acids.
with several 5-mL portions of hexane, and the organic phases TMS ether derivatives of alcohols have been widely used
were combined and dried on anhydrous sodium sulfate. After for identification purposes because of their high volatility,
filtering, the solvent was stripped off and the residue was which makes them particularly useful for GC separation
taken up in hexane and analyzed by GC–MS.
of nonvolatile products, and the characteristic α-cleavage
GC–MS analyses were run on a Hewlett-Packard 5989 A products of high diagnostic value they induce (17). Derivati-
mass spectrometer coupled to a Hewlett-Packard 5890 series zation, as the TMS ethers, showed the presence of a third com-
II-gas chromatograph (Hewlett-Packard, Palo Alto, CA) in ponent by GC–MS (Fig. 1B), probably an isomer of the other
electron impact (EI) and chemical ionization (CI) mode. CI two esters according to their very similar chromatographic
used methane as the ionizing gas. Derivatized compounds as features. An improved resolution of a mixture of dihy-
TMS derivatives were separated on a methyl silicone fused- droxyoctadecanoic acids through derivatization as the TMS
silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film derivatives was also noticed by Huang et al. (18). EI
thickness). The temperature program was injection at 60ºC, mass spectra of the TMS derivatives showed that the first-
increased at 15ºC/min up to 250ºC, and held at this tempera- eluting compound (GC Rt = 13.723 min) presented a high-
JAOCS, Vol. 78, no. 6 (2001)

PRODUCTION OF (E)-11-OH-9- AND (E )-9-OH-10-OCTADECENOIC ACIDS
595
FIG. 1. (A) Gas chromatogram of the methyl esters of monohydroxy octadecenoic acids 1 (elut-
ing at a retention time of 13.723 min) and 2–3 (coeluting at a retention time of 14.983 min)
obtained by oxidation of oleic acid with Pseudomonas sp. 32T3. (B) Selected ion monitoring
mass spectra of ions of m/z 227, 271, and 285 of the corresponding trimethylsilyl derivatives.
intensity fragment at m/z 285, which was attributed to the ion hydroxy-8-octadecenoic acid, and (E)-9-hydroxy-10-octade-
[Me3SiOCHCH=CH(CH2)7COOCH3]+, indicating that the cenoic. Location of the double bond in each compound was
hydroxyl group was at position 11 (Fig. 2A). The spectra determined on the basis of its NMR spectrum (see below).
of the other two compounds, which coeluted at a GC Rt
The purified extract was subjected to CI–MS analysis. The
of 14.983 min, showed the two largest fragment ions of m/z spectra of the TMS derivatives provided evidence for the
271 [Me3SiOCHCH=CH(CH2)6COOCH3]+ and m/z 227 presence of monohydroxy octadecenoate derivatives of mo-
[Me3SiOCHCH=CH(CH2)6CH3]+, arising from α cleavage of lecular weight 384 with a base peak of m/z 295 [M −
the silylated hydroxyl group (Fig. 2B). No molecular ion was OTMS]+. Other important diagnostic ions were the fragments
apparent from the spectra. The fragment ion of m/z 271 sug- of m/z 369 [M − CH3]+, 263 [M − OTMS − CH3OH]+, 383
gested the presence of a hydroxyl group at C10 while that of [M − H]+, 385 [M + H+], as well as the α cleavage fragments
m/z 227 is consistent with a C8–C9 bond cleavage of a fatty of m/z 285, 271, and 201 (see above).
acid with the hydroxyl group localized at C9. The other ex-
The 1H NMR spectrum of the hydroxyester fraction
pected ions of m/z 201, 215 and 259, corresponding to α cleav- showed in the olefinic region the presence of a doublet of
age of the TMS ether, were not apparent from the spectra, most triplets at δ 5.621 (J = 15.0 Hz, J′ = 6.6 Hz) corresponding to
likely because they represent the unlikely breakage of a vinylic Hb of the HOCHCHa=CHb group and a doublet of doublet of
bond (Fig. 2). According to these data, the compounds were triplets centered at δ 5.444 (J = 15.3 Hz, J′ = 7.0 Hz, J″ = 1.1
postulated to be (E)-11-hydroxy-9-octadecenoic acid, (E)-10- Hz) corresponding to Ha. The vinylic coupling constant
JAOCS, Vol. 78, no. 6 (2001)

596
E. RODRÍGUEZ ET AL.
FIG. 2. (A) Electron impact (EI) mass spectrum and origin of ion of m/z 285 of the trimethylsilyl
derivative of the methyl ester of (E)-11-hydroxy-9-octadecenoic acid (1). (B) EI mass spectrum
and origin of ions of m/z 271 and 227 of the trimethylsilyl derivatives of the methyl esters of
(E)-10-hydroxy-8-octadecenoic acid (2) and (E)-9-hydroxy-10-octadecenoic acid (3).
the 13C NMR spectrum the following diagnostic signals were
apparent: 174.31 ppm (CO), 131.9–133.2 ppm (cluster of
olefinic carbons), 77.29 and 73.24 (CHOH), and 51.4
(OCH3). These data point to the presence of (E)-11-hydroxy-
9-octadecenoic acid (1), (E)-10-hydroxy-8-octadecenoic acid
(2), and (E)-9-hydroxy-10-octadecenoic acid (3), the first and
third compounds being chemicals arising from bioconversion
JHa-Hb of 15.0–15.3 Hz indicated that the stereochemistry of
the double bond was trans. Other key signals of the spectrum
were a doublet of triplets at δ 4.03 with coupling constants
J = 6.6 Hz, J′ = 6.3 Hz, corresponding to the methine proton
of an allylic hydroxyl group (HOCHC=C), a triplet at δ 2.30
(J = 7.4 Hz) of a methylene group in α position to the car-
bonyl, and a multiplet at δ 2.02 of the allylic methylene(s). In
JAOCS, Vol. 78, no. 6 (2001)

PRODUCTION OF (E)-11-OH-9- AND (E )-9-OH-10-OCTADECENOIC ACIDS
597
by Bacillus Strain NRRL BD-447: Identification of 7-Hydroxy-
17-oxo-9-cis-octadecenoic Acid, J. Am. Oil Chem. Soc. 76:
1023–1026 (1999).
of edible waste oil. Compound 1 may result from direct al-
lylic oxidation of oleic acid at C-11, and in fact it was ob-
tained by Knothe et al. (19) by chemical oxidation of oleic
acid with SeO2/tert-butylhydroperoxide. Compound 2 is a
fungitoxic chemical isolated from the timothy plant Epichloe
typhina (20) and was identified in the products of the action
of Pseudomonas sp. 42A2 on oleic acid (11), and by Kim
et al. (15) in the products of the action of P. aeruginosa on
oleic acid. Compound 3, reported by Koshino et al. (20), may
well proceed from the oxidation of oleic acid at C-9 and mi-
gration of the double bond to C-11. Allylic hydroxylations
with concomitant migration of double bonds are known to
occur in the cytochrome P-450 oxidation of fatty acids (21).
10. Parra, J.L., J. Pastor, F. Comelles, A. Manresa, and M.P. Bosch,
Studies of Biosurfactant Obtained from Olive Oil, Tenside Surf.
Det. 27:302–306 (1990).
11. 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
Pseudomonas sp. 42A2, Biochim. Biophys. Acta 1347:75–81
(1997).
12. Bastida, J., C. de Andrés, J. Culleré, M. Busquets, and A. Man-
resa, Biotransformation of Oleic Acid into 10-Hydroxy-8E-oc-
tadecenoic Acid by Pseudomonas sp. 42A2, Biotechnol. Lett.
21:1031–1035 (1999).
13. Hou, C.T., M.O. Bagby, R.D. Plattner, and S. Koritala, A Novel
Compound, 7,10-Dihydroxy-8(E)-octadecenoic Acid from
Oleic Acid by Bioconversion, J. Am. Oil Chem. Soc. 68:99–101
(1991).
ACKNOWLEDGMENTS
We gratefully acknowledge CICYT (projects PPQ 2000-0105-P4-
03 and AGL 20000-1695-C02-01), EC (FAIR CT96-1302), and
Comissionat per a Universitats i Recerca (1997SGR 00021 and
1999SGR00024) for financial support.
14. Gardner, H.W., and C.T. Hou, All (S) Stereoconfiguration of
7,10-Dihydroxy-8-(E)-octadecenoic Acid from Bioconversion
of Oleic Acid by Pseudomonas aeruginosa, Ibid. 76:1151–1156
(1999).
15. Kim, H., H.W. Gardner, and C.T. Hou, 10(S)-Hydroxy-8(E)-
octadecenoic Acid, an Intermediate in the Conversion of Oleic
Acid to 7,10-Dihydroxy-8(E)-octadecenoic Acid, Ibid. 77:
95–99 (2000).
16. Moore, J.A., and D.E. Reed, Diazomethane, in Organic Synthe-
sis, edited by V. Baumgarten, John Wiley & Sons, New York,
Vol. V, 1973, pp. 351–355.
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