Chemoenzymatic production of (+)-coriolic acid from trilinolein: Coupled synthesis and extraction

Article abstract of DOI:10.1007/s11746-997-0196-8

Chemoenzymatic conversion of trilinolein to (+)-coriolic acid was investigated in this work. Lipase-catalyzed hydrolysis of trilinolein and lipoxygenation of liberated linoleic acid were coupled in a two-phase medium that consisted of a pH 9 borate buffer and a water-immiscible organic solvent (octane). High concentrations of trilinolein could be dissolved in the organic phase (up to 340 mM). Linoleic acid, liberated after hydrolysis, transferred to the aqueous phase and was enzymatically converted to the preferred 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid with soybean lipoxygenase-1. This product, which remained in the aqueous phase, could be recovered by centrifugation and then chemically reduced to (+)-coriolic acid (purity >95%). Recovery of this compound by liquid-liquid extraction was easy. The structure of (+)-coriolic acid has been confirmed by ¹H nuclear magnetic resonance spectroscopy, mass spectrometry, and infrared spectroscopy. High yields were obtained with pure trilinolein or sunflower oil as initial substrates.

Full text of DOI:10.1007/s11746-997-0196-8

Chemoenzymatic Production of (+)-Coriolic Acid
from Trilinolein: Coupled Synthesis and Extraction
Mohamed Gargouri* and Marie Dominique Legoy
Laboratoire de Génie Protéique et Cellulaire, Pôle Sciences et Technologie,
Université de La Rochelle, 17042 La Rochelle Cédex 1, France
ABSTRACT: Chemoenzymatic conversion of trilinolein to
In plants, HPOD is an intermediate compound in the LOX
pathway (11,12). Lipase (E.C.3.1.1.3)-catalyzed hydrolysis
of trilinolein (TL) produces LA, which is oxygenated in the
presence of LOX (E.C.1.13.11.12) and molecular oxygen.
The HPOD obtained are highly reactive compounds (13).
The majority of intracellular enzymes use the products of
other enzymes as substrates, and the biocatalysis does not
take place in a homogeneous medium but rather in a compart-
mentalized cellular structure. A number of multienzymatic
systems, consisting essentially of membrane- (14) or matrix-
(15,16) bound enzymes, have been studied. These enzymes
(+)-coriolic acid was investigated in this work. Lipase-catalyzed
hydrolysis of trilinolein and lipoxygenation of liberated linoleic
acid were coupled in a two-phase medium that consisted of a
pH 9 borate buffer and a water-immiscible organic solvent (oc-
tane). High concentrations of trilinolein could be dissolved in
the organic phase (up to 340 mM). Linoleic acid, liberated after
hydrolysis, transferred to the aqueous phase and was enzymati-
cally converted to the preferred 13(S)-hydroperoxy-9Z,11E-
octadecadienoic acid with soybean lipoxygenase-1. This prod-
uct, which remained in the aqueous phase, could be recovered
by centrifugation and then chemically reduced to (+)-coriolic
acid (purity >95%). Recovery of this compound by liquid–liq- catalyze a set of consecutive chemical reactions and are then
uid extraction was easy. The structure of (+)-coriolic acid has
been confirmed by 1H nuclear magnetic resonance spec-
troscopy, mass spectrometry, and infrared spectroscopy. High
yields were obtained with pure trilinolein or sunflower oil as
initial substrates.
close to their respective substrates.
Bienzymatic reactions have been studied in low-water
media and show interesting aspects regarding the transfer and
partition of reaction components (17). Recently (18), we have
studied a bienzymatic system that consisted of lipase and
LOX in a water-organic biphasic medium. Polyunsaturated
fatty acid triacylglycerols were converted to HPOD with high
yields.
In this work, we present a chemoenzymatic conversion of
TL to (+)-coriolic acid in two steps. The use of TL as start-
ing substrate is a new approach. The first step is enzymatic
and consists of coupled hydrolysis and lipoxygenation in a
JAOCS 74, 641–645 (1997).
KEY WORDS: Bienzymatic bioreactor, biphasic media,
chemoenzymatic synthesis, (+)-coriolic acid, hydroperoxide,
linoleic acid, liquid–liquid extraction, Pseudomonas sp. lipase,
soybean lipoxygenase-1, trilinolein.
(+)-Coriolic acid (13S-hydroxy-9Z,11E-octadecadienoic two-phase medium (borate buffer/octane). The first substrate
acid) is a metabolite of the lipoxygenase (LOX) pathway of (TL) is dissolved in octane and is hydrolyzed in the presence
linoleic acid (LA). This acid plays a physiological role in of lipase at the octane-water interface. The produced free
plant and animal cells. It is present in heart mitochondria (1) fatty acid, being poorly water-soluble, transfers slowly to the
and takes part in the defense against rice blast disease (2) and aqueous phase, which contains the LOX (19). Soybean
in the synthesis mechanism of prostacyclin PGI2 (3). It has lipoxygenase-1 (LOX-1), used in this work, has the ad-
also been shown to act as a chemorepellant that influences vantage of high selectivity. It converts LA preferentially
platelets, leukocytes, or malignant cells (4–6).
to 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13S-
The synthesis of (+)-coriolic acid has been already HPOD) (20). The lipoxygenation takes place in the bulk of
achieved chemically (7,8) and chemoenzymatically (9). Mar- the aqueous phase. The produced HPOD is hydrophilic and
tini et al. (10) have produced (+)-coriolic acid from LA by therefore remains in the aqueous phase (21). The kinetic be-
lipoxygenation under oxygen pressure (2.5 bar), followed by havior of this bienzymatic system in a two-phase bioreactor
reduction of the hydroperoxide (HPOD).
was previously studied (22). Interactions between biocon-
version and mass transfer were observed. In the second step,
the aqueous phase is separated from the organic phase. 13S-
HPOD is then chemically reduced to (+)-coriolic acid
(Scheme 1).
*To whom correspondence should be addressed at Laboratoire de Génie
Protéique et Cellulaire, Pôle Sciences et Technologie, Université de La
Rochelle, Avenue Marillac, 17042 La Rochelle Cédex 1, France.
E-mail: mgargour@bio.univ-lr.fr.
Copyright © 1997 by AOCS Press
641
JAOCS, Vol. 74, no. 6 (1997)

642
M. GARGOURI AND M.D. LEGOY
Chemical reduction of HPOD and extraction of hydroxy
acids (HOD). At the end of the enzymatic step, the organic
and aqueous phases were separated and proteins were elimi-
nated with centrifugation. The aqueous phase, containing all
the HPOD (98–99%) produced in the enzymatic step, was
added to 2 mL ethanol and NaBH4 (1 mg for 4 mg of initial
TL). After 1 h at 25°C, the reaction was stopped by adding
1 M HCl. HOD was then extracted with diethyl ether (3× the
same volume). The organic phase was dried with MgSO4, and
diethyl ether was evaporated under vacuum. HOD was di-
luted in ethanol and stored at −25°C.
Analysis. Separation and analysis of the different acylglyc-
erols and fatty acids were carried out by HPLC equipped with
an ultraviolet (UV) detector (Waters 486 at 210 nm, Paris,
France) and a C18 reverse-phase column (µBondapak 3.9 mm
× 300 mm; Waters). Samples (10 µL) were periodically with-
drawn from the two-enzyme reactor and diluted 10 to 100
times before analysis. The mobile phase had the following
composition: acetonitrile/acetone/acetic acid (7:4:0.02,
vol/vol/vol). The pump rate was 1.8 mL/min.
Measurements of HPOD and HOD concentrations were
performed at 234 nm on a Perkin-Elmer UV (Lambda16)
spectrophotometer, assuming a molar extinction coefficient
value of ε = 25,000 cm−1·M−1 for the two compounds. Sample
withdrawal has been described elsewhere (19).
Regioselectivities of HOD and HPOD were analyzed by
HPLC (UV detection at 234 nm) with a Hypersil-10 µ San-
don (Paris, France) silica column (4.6 mm × 300 mm). The
elution phase (1.5 mL/min) was composed of hexane/diethyl
ether/acetic acid (7.5:2.5:0.01, vol/vol/vol). Samples with-
drawn from the reduction medium were diluted in water. To
stop the reaction, 1 M HCl was added at 0°C.
Trilinolein
Enzyme 1
Enzyme1
LIPASE
LIPASE
COOH
Polyunsaturated fatty acid
[linoleic acid (9Z,12Z)]
[Linoleic acid (9Z,12Z)]
Polyunsaturated fatty acid
Enzyme 2
Enzyme2
LIPOXYGENASE, O
LIPOXYGENASE, O2
2
Hydroperoxy acid
Hydroperoxy-acid
[13S-hydroperoxy-9Z,11E-octadecadienoic acid]
[13S-hydroperoxy-9Z,11E-octadecadienoic acid]
COOH
OOH
OOH
REDUCTION
REDUCTION
Hydroxy-acid
Hydroxy acid
[13S-hydroxy-9Z,11E-octadecadienoicacid]
[13S-hydroxy-9Z,11E-octadecadienoic acid]
[(+)-Coriolic acid]
[(+)-coriolic acid]
COOH
OH
OH
SCHEME 1
EXPERIMENTAL PROCEDURES
Purification and characterization of (+)-coriolic acid. (+)-
Coriolic acid was separated from the other isomers by liquid
chromatography (20 mm × 300 mm) over silica gel 60
(Merck, Paris, France) with 2% (10 min) to 4% (increasing
0.25% per min) methanol in dichloromethane (vol/vol; 4
mL/min). Pure (+)-coriolic acid was then obtained (45 mg) as
a colorless oil. Found: m/z 296.2346; C18H32O3 requires M+,
296.2351; νmax (cm−1), 3300, 3055, 2928, 2855, 1730, 1440,
1422, 995, and 896:
Materials. LA, TL, and LOX-1 type 1-S (60% protein) were
purchased from Sigma Chemical Co. (Paris, France). Sol-
vents [high-performance liquid chromatography (HPLC)
grade] were obtained from Aldrich (Paris, France). Crude
powder lipase from Pseudomonas sp. (LPS) was kindly pro-
vided by Amano (Nagoya, Japan). Enzymes were used with-
out further purification. Water was MilliQ grade (18.2
MΩcm; Millipore, Paris, France). Sunflower oil was obtained
from Huilerie Lezay (Région Poitou-Charentes, France). In-
frared (IR) spectra were recorded on a Perkin-Elmer Paragon
1000PC instrument (Paris, France). 1H nuclear magnetic res-
onance (NMR) spectra were recorded on a Bruker
AM300WB spectrometer (Paris, France). Mass spectra were
realized by “Centre Régional de Mesures Physiques de
l’Ouest, Université de Rennes 1.”
Coupling of LPS and LOX-1. Coupled hydrolysis and
lipoxygenation were carried out in 50-mL flasks with a work-
ing volume of 12 mL (2 mL octane and 10 mL borate buffer,
pH 9). Flasks were incubated in rotary shakers at 250 rpm
under constant temperature control (18). TL was initially dis-
solved in the organic phase, and enzymes were dissolved in
the aqueous phase. Oxygen (30 mL/min) was continuously
flushed through the medium. Typical reaction times were of
the order of 5 h.
10
18
12
13
9
16 14
15
7
5
3
COOH
1
4
6
2
11
17
8
OH
1
δ H (300 MHz, CDCl3): 0.85 (3H, t, J = 8 Hz, 18-CH3),
1.20–1.35 (14H, m, 17, 16, 15, 7, 6, 5, 4-CH2), 1.50–1.65 (4H,
br m, 14, 3-CH2), 2.15 (2H, m, 8-CH2), 2.35 (2H, t, J = 9 Hz,
2-CH2), 4.20 (1H, m, 13-CH), 5.42 (1H, dt, J1 = 7 Hz, J2 = 11
Hz, 9-CH), 5.65 (1H, dd, J1 = 7 Hz, J2 = 15Hz, 12-CH), 5.95
(1H, t, J1 = J2 = 11 Hz, 10-CH) and 6.47 (1H, dd, J1 = 14 Hz,
J2 = 11 Hz, 11-CH); reference peaks: TMS, m/z 296 (M+,
3%), 278 (M+ − H2O, 11), 236 (2), 221 (1), 207 (10), 189 (2),
179 (7), 164 (6), 147 (7), 135 (12), 125 (4), 121 (9), 109 (11),
and 99 (100).
JAOCS, Vol. 74, no. 6 (1997)

CHEMOENZYMATIC SYNTHESIS OF (+)-CORIOLIC ACID
643
TABLE 1
100
80
60
40
20
0
Yields (mM) of the Different Steps of Hydroxy Acid (HOD)
Preparation from Trilinolein (TL) for Chemoenzymatic
Synthesis Followed by Liquid–Liquid Extractiona
LA/TL HPOD/LA HPOD/TL
HOD/TL HODext/TL
TL (g/L)
(%)
(%)
(%)
(%)
(%)
20
50
70
100
120
150
200
300
95.1
90.5
89.4
87.9
79.8
79.4
47.5
44.2
91.6
80.6
64.7
56.3
55.8
52.7
55.2
55.7
87.1
72.9
57.8
49.5
44.5
41.8
26.2
24.6
87.1
72.9
57.8
49.5
44.5
41.8
26.2
24.6
78.4
62.7
50
36.9
33.9
29
18.5
17.2
aConditions: 1.2 mg crude powder lipase from Pseudomonas sp. (LPS) and
0.25 mg soybean lipoxygenase-1 (LOX-1) were used per 1 mg TL at 28°C.
Conversion of TL to linoleic acid (LA) (hydrolysis yield = LA/TL) was mea-
sured by reverse-phase high-performance liquid chromatography (HPLC).
Syntheses of hydroperoxide (HPOD) and HOD were measured spectropho-
tometrically after 5 h. Lipoxygenation yield = HPOD/LA, and hydroly-
sis–lipoxygenation yield = HPOD/TL. Chemoenzymatic synthesis of HOD
from TL before (hydrolysis–lipoxygenation–reduction yield = HOD/TL) and
after extraction (total yield = HODext/TL) was confirmed by HPLC (silica).
0
10
20
LPS (g/L)
[LPS] (g/L)
FIG. 1. Dependence of the yield of hydroperoxide (mM)/trilinolein (mM)
RESULTS
on the concentration crude powder lipase from Pseudomonas sp. in the
aqueous phase at 28°C. The same conditions were used as in Table 1.
Two-enzyme reactor. The yields of hydrolysis and lipoxy-
genation, HPOD (mM)/TL (mM), were studied as a function
of temperature. The initial concentration of TL was 50 g/L in
octane. Concentrations of LPS and LOX-1 in the aqueous
phase were, respectively, 12 and 25 g/L. The reaction period
was 5 h with a constant flow of O2 (30 mL/min). The optimal
temperature for the bienzymatic system LPS/LOX-1 was
28°C (yield = 72.9%). The yield changed significantly at ei-
ther side of the optimal temperature (yield = 62% at 26°C and
51.5% at 30°C).
TABLE 2
Yields of the Different Steps of HOD Preparation from Crude
Sunflower Oil by the Chemoenzymatic Synthesis Followed
by Liquid–Liquid Extractiona
LA/TL HPOD/LA HPOD/oil HPOD/TL HOD/TL HODext/TL
TL (g/L) (%)
(%)
(%)
(%)
(%)
(%)
20
50
100
200
89
69.2
70
89
90.5
75
47.5
37.6
31.5
21.2
79.2
62.6
52.5
35.3
79.2
62.6
52.5
35.3
70
52.6
39.5
25
47
75.1
In the biphasic medium, crude powder LPS and LOX-1
were coupled by using different lipase concentrations. Five
concentrations of LPS were tested. The yields of HPOD/TL
are presented in Figure 1. These results indicate that the yield
of HPOD synthesized from TL is better when an LPS concen-
tration of 12 g/L is used in the aqueous phase for 50 g/L TL
concentration in the organic phase. Higher concentrations of
LPS enhance the yield only slightly. Logically, we chose 1.2
mg LPS for 1 mg TL for the following experiments in the
biphasic system (ratio octane/borate buffer = 1:5).
Preparation of (+)-coriolic acid from TL. Intermediaries
and final yields of the chemoenzymatic synthesis of HOD
from TL were studied as a function of initial TL concentra-
tion (Table 1). High yields were obtained especially when
using concentrations of TL below 100 g/L in the organic
phase of the biphasic medium. The yield of extracted product
decreased when the initial TL concentration was increased.
The chemical reduction of HPOD to HOD is complete for all
concentrations.
aThe same conditions as those in Table 1 were used. For abbreviations, see
Table 1; HPOD/oil means yield of the bienzymatic system in relation to sun-
flower oil (%).
ble quantities (<0.02%) in the sunflower oil. This oil contains
6% palmitic acid, 4.5% stearic acid, and 20% oleic acid,
which are not substrates of LOX-1. In the two-enzyme sys-
tem, lipolysis of triglycerides liberates fatty acids, which
transfer to the aqueous phase. Only LA, the single polyunsat-
urated fatty acid in sunflower oil, was oxidized in the pres-
ence of LOX. HPOD obtained in the bienzymatic reactor
were reduced chemically. Yields were studied as a function
of the initial oil concentration (Table 2). They were calculated
in relation to TL present in the sunflower oil after every step.
A second yield for the two-enzyme system was calculated in
relation to the total sunflower oil (HPOD/oil). These yields
approach the yields obtained with the pure TL as substrate.
Regioselectivity. HPLC analysis of HPOD produced in the
two-enzyme system shows one major peak (94%), which cor-
responds to the 13S-HPOD as in an aqueous medium.
Chemoenzymatic synthesis of HOD from TL or sunflower oil
does not change the regioselectivity (Table 3).
Preparation of (+)-coriolic acid from a crude vegetable
oil. In the next section of work described here, we used a
crude vegetable oil rich in LA (sunflower oil) rather than pure
TL. This allows a more realistic scenario for industrial-scale
manufacture of HOD and was the main reason for developing
this chemoenzymatic approach. LA is the major fatty acid
component (60% molar). Linolenic acid is present in negligi-
DISCUSSION
JAOCS, Vol. 74, no. 6 (1997)

644
M. GARGOURI AND M.D. LEGOY
TABLE 3
is interesting because of the near total recovery of the product
by simple centrifugation. Thus, the aqueous phase, containing
HPOD, can be separated from the organic phase and solid pro-
tein. Chemical reduction of HPOD in the presence of alcohol
was complete. HOD then was extracted and stored in ethanol.
The regioselectivity obtained is similar to the regioselectivity
of HPOD. The single major compound is (+)-coriolic acid
(>95%), product of the reduction of 13S-HPOD (major isomer
of HPOD obtained in our system). The structure of (+)-cori-
olic acid was confirmed by 1H NMR, MS, and IR.
Regioselectivity (%) of HPOD and HOD in Aqueous Mediuma pH 9b
After Chemoenzymatic Synthesis from TLc or Sunflower Oild
as Substrates
13(S)9Z,11E
13(S)9E,11E
9(S)10E,12Z
9(S)10E,12E
HPODb
HPODc
HODc
95
94
96.2
96
0.5
0.5
0.4
0.6
2.7
3.3
2.9
2.5
1.8
2.2
0.5
0.9
HODd
aExperimental conditions are those in Table 1, where abbreviations can be
found.
bYield in aqueous medium, pH 9.
cSubstrate: TL.
Evolution of HPOD in the two-enzyme system had an op-
timal rate between 150 and 200 min. At 300 min, production
of HPOD became much slower because lipoxygenase was in-
activated at the octane/borate buffer interface (19). In the re-
dSubstrate: sunflower oil.
We have studied the synthesis of (+)-coriolic acid by a duction medium, HPOD was rapidly converted to HOD (95%
chemoenzymatic method. Three reactions were necessary to in 20 min). After one hour, the reduction was complete.
convert TL to (+)-coriolic acid. In the first step, lipase-cat-
Yields were also high when using crude sunflower oil
alyzed hydrolysis of TL and lipoxygenation of liberated LA (60% LA) as the initial substrate. The quality of the final
were coupled in a biphasic system. Presence of an organic product [(+)-coriolic acid] after synthesis and extraction was
phase (octane) in the medium allows high solubility of acyl- the same as that obtained with TL as substrate. In fact, after
glycerols and fatty acids. High substrate concentrations were hydrolysis of triglycerides, only LA was oxygenated in the
then used. In the biphasic medium, mass transfer across the presence of LOX-1; the medium did not contain other polyun-
liquid–liquid interface is controlled. The moderately alkaline saturated fatty acids. After production of HPOD in the two-
pH (9) facilitates LA solubilization and results in a sodium enzyme system, the aqueous phase was separated from the or-
salt being formed. Recently (19), we showed that the sub- ganic phase and enzyme. HPOD was then separated from im-
strate (LA) and the product (HPOD) of the LOX reaction purities existing in the sunflower oil. The final extraction after
have surface-active properties. During lipoxygenation in the synthesis of (+)-coriolic acid offered a high degree of purity.
biphasic system, the consumption of LA and the production
The approach used in this work could be easily extended
of HPOD favor the fatty acid transfer to the aqueous phase, to a large scale to produce (+)-coriolic acid chemoenzymati-
which allows an increase in LOX reaction. In the two-enzyme cally from high concentrations of TL with facile extraction.
system (LPS/LOX-1), the phenomenon described previously
favors lipoxygenation of LA. The consumption of this inter-
mediate allows an increase in the degree of LPS reaction at
This work was supported by a grant from “La Région Poitou-Char-
the liquid–liquid interface. These interactions between the
two reactions and the mass transfer increase the yields ob-
tained in the two-enzyme reactor.
ACKNOWLEDGMENTS
entes,” France. The authors are grateful to Dr. Marie Claire Parker
for help with correction of the text.
The optimal temperature for the production of HPOD from
TL in the biphasic medium was 28°C and allows high yields
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