Stereospecific production of 9R-hydroxy-10E,12Z-octadecadienoic acid from linoleic acid by recombinant Escherichia coli cells expressing 9R-lipoxygenase from Nostoc sp. SAG 25.82

Article abstract of DOI:10.1016/j.molcatb.2014.03.009

One of the most significant properties of lipoxygenase (LOX) as a biocatalyst is its stereo-selective oxygenation. In this study, the stereo-specific production of 9R-hydroxy-10E,12Z-octadecadienoic acid (9R-HODE) from linoleic acid was achieved using whole recombinant Escherichia coli cells expressing LOX from Nostoc sp. SAG 25.82. The optimal conditions for the production of 9R-HODE were pH 7.5, 25 °C, 40 g l^-1 cells, 15 g l^-1 linoleic acid, 2% (v/v) methanol, 1 working volume/oxygen volume/min (vvm) oxygenation rate, and 250 rpm agitation speed in 500 ml-baffled flask containing a working volume of 50 ml. Under these optimized conditions, whole recombinant cells expressing 9R-LOX protein produced 14.3 g l^-1 9R-HODE for 1 h, with a conversion yield of 95% (w/w) and a productivity of 14.3 g l^-1 h^-1. The oxygen supply method significantly influenced stereo- and regio-selectivity of the oxygenation of linoleic acid. Among the oxygen supply methods tested, oxygenation (1 vvm) with agitation (250 rpm) resulted in the highest 9R/13S-HODE ratio of the products at 96:4. This is the first application using whole recombinant cells harboring R-specific LOX for the stereo-selective production of an R-specific hydroxy fatty acid.

Full text of DOI:10.1016/j.molcatb.2014.03.009

Journal of Molecular Catalysis B: Enzymatic 104 (2014) 56–63
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Stereospecific production of 9R-hydroxy-10E,12Z-octadecadienoic
acid from linoleic acid by recombinant Escherichia coli cells expressing
9R-lipoxygenase from Nostoc sp. SAG 25.82
Kyoung-Rok Kima, Min-Ho Seoa, Jin-Byung Parkb, Deok-Kun Oha,∗
a Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea
b Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 January 2014
Received in revised form 24 February 2014
Accepted 13 March 2014
Available online 24 March 2014
One of the most significant properties of lipoxygenase (LOX) as a biocatalyst is its stereo-selective
oxygenation. In this study, the stereo-specific production of 9R-hydroxy-10E,12Z-octadecadienoic acid
(9R-HODE) from linoleic acid was achieved using whole recombinant Escherichia coli cells expressing
LOX from Nostoc sp. SAG 25.82. The optimal conditions for the production of 9R-HODE were pH 7.5,
25 ◦C, 40 g l−1 cells, 15 g l−1 linoleic acid, 2% (v/v) methanol, 1 working volume/oxygen volume/min (vvm)
oxygenation rate, and 250 rpm agitation speed in 500 ml-baffled flask containing a working volume of
50 ml. Under these optimized conditions, whole recombinant cells expressing 9R-LOX protein produced
14.3 g l−1 9R-HODE for 1 h, with a conversion yield of 95% (w/w) and a productivity of 14.3 g l−1 h−1. The
oxygen supply method significantly influenced stereo- and regio-selectivity of the oxygenation of linoleic
acid. Among the oxygen supply methods tested, oxygenation (1 vvm) with agitation (250 rpm) resulted
in the highest 9R/13S-HODE ratio of the products at 96:4. This is the first application using whole recom-
binant cells harboring R-specific LOX for the stereo-selective production of an R-specific hydroxy fatty
acid.
Keywords:
9R-Hydroxy-10E,12Z-octadecadienoic acid
Lipoxygenase
Nostoc sp. SAG 25.82
Oxygen supply
Stereo-selective oxygenation
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
fatty acids [6–8]. HFAs have been found in bacterial, fungal, and
mammalian lipids not only as signaling molecules, but also as bioac-
Lipoxygenase (LOX, EC 1.13.11.-) is a non-heme iron-containing
dioxygenase, which catalyzes the oxidation of polyunsaturated
fatty acids (PUFAs) with one or several (1Z,4Z)-pentadiene moi-
eties to produce specific E,Z-conjugated-hydroperoxy PUFAs and
their corresponding reduced hydroxy fatty acids (HFAs) [1,2]. These
fatty acids are subsequently metabolized into important bioactive
signaling compounds, such as leukotrienes and lipoxins in animals
[3] and jasmonic acids in plants [4]. LOXs have been extensively
studied in animals and plants, however, the existence of LOXs in
coral, moss, algae, fungi, and bacteria has been recently reported
[5].
HFAs are used as starting materials for the synthesis of poly-
mers and additives in the manufacturing of lubricants, emulsifiers,
and stabilizers in the chemical, food, cosmetic, and pharmaceu-
tical industries because they possess higher reactivity, solvent
miscibility, stability, and viscosity, compared to non-hydroxylated
tive compounds, such as antibacterial, antifungal, and anticancer
agents [7]. Notably, the stereo-specific HFA 9R-hydroxy-10E,12Z-
octadecadienoic acid (9R-HODE) is a known signaling molecule that
directly binds to the human G-protein-coupled receptor [9]. 9R-
HODE exhibits fungicidal and fat degradation activity and induces
interleukin-1 beta expression [10,11]. The first reaction for the syn-
thesis of fatty acid-derived bioactive compounds is the insertion of
molecular oxygen into unsaturated fatty acid by cytochrome P450
and LOX. However, these enzymes have not been used as biocata-
lysts because of the difficulties associated in achieving high-level
production of bioactive compounds and high-level expression in
hosts. For the use of these enzymes as biocatalysts, the stereo-
specific production of HFAs is essential.
LOXs have mainly been applied to the production of hydroper-
oxy fatty acids (HPFAs) instead of HFAs. For example, soybean
LOXs, both free and immobilized enzymes in water and solvent
systems (mono-, di-, and multi-phasic solvents), have been exten-
sively utilized to produce 13-hydroperoxy octadecadienoic acid
(13-HPODE) from linoleic acid and oils [12–20]. Bacterial [21]
and fungal [22] LOXs produce 13-HPODE from linoleic acid, and
∗
Corresponding author. Tel.: +82 2 450 4118; fax: +82 2 444 5518.
E-mail address: deokkun@konkuk.ac.kr (D.-K. Oh).
http://dx.doi.org/10.1016/j.molcatb.2014.03.009
1381-1177/© 2014 Elsevier B.V. All rights reserved.

K.-R. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 56–63
57
potato LOX produces 9S-HPODE [15]. The production of HFAs has
been reported in LOXs from only several sources, such as potato
[23,24], fungi [25], and cyanobacteria [26]. Potato and fungal LOXs
produce 9S-specific HFAs, whereas the cyanobacteria Anabaena
flos-aquae f. flos-aquae and Anabaena sp. PCC 7120 produces
9R-specific HFAs [26,27], suggesting that cyanobacterial LOXs
exhibit unusual stereo-selectivity. However, the biotechnological
production of 9R-specific HFAs using R-specific LOXs has not been
attempted.
E. coli ER2566 cells by electroporation. The expression of the 9R-
LOX gene was induced using 500 ml of LB medium in a 2000 ml
flask containing 50 ␮g ml−1 ampicillin. To induce enzyme expres-
sion, isopropyl-␤-d-thiogalactopyranoside (IPTG) was added to a
final concentration of 0.1 mM at the optical density of the bacteria
of 0.6 at 600 nm, and the culture incubated for an additional 16 h
at 16 ◦C [31].
2.4. Effects of pH, temperature, and thermostability
In the present study, the stereo-selective biocatalyst 9R-LOX
from Nostoc sp. was cloned and expressed in Escherichia coli to
produce 9R-HODE from linoleic acid. The reaction conditions for
the production of 9R-HODE via whole recombinant cells, includ-
ing organic solvent, pH, temperature, oxygen supply method, and
the concentrations of substrate, were optimized. Under the opti-
mized conditions, the stereo-specific production of 9R-HODE was
achieved.
Unless otherwise stated, reactions were performed at 25 ◦C
for 20 min in 50 mM Tris–HCl buffer (pH 7.5) containing 10 g l−1
cells and 2 g l−1 linoleic acid. To examine the effect of pH on the
production of 9R-HODE by whole recombinant cells expressing
9R-LOX from Nostoc sp., the pH was varied from 6.5 to 8.5 using
100 mM sodium phosphate buffer (pH 6.5–8.0) and 50 mM Tris–HCl
buffer (pH 7.0–8.5). The effect of temperature on the production of
9R-HODE by whole recombinant cells expressing 9R-LOX was eval-
uated by varying the temperature from 10 to 45 ◦C. The stability of
whole recombinant cells was measured after incubation for 3.5 h
under the standard reaction conditions as described above. Sam-
ples were withdrawn at several time intervals and the activity for
the production of 9R-HODE was determined.
2. Materials and methods
2.1. Chemicals
The 9R-, 9S-, 13S-HODE, and 9-HPODE standards were pur-
chased from Cayman Chemical (98%; Ann Arbor, MI, USA).
The linoleic acid standard and SnCl2 were purchased from
Sigma-Aldrich (>99%; St. Louis, MO, USA). The linoleic acid
substrate was purchased from TCI (>95%; Tokyo, Japan).
Tris(hydroxymethyl)aminomethane (Tris), NaH2PO4, Na2HPO4,
and sodium NaBH4 were purchased from Daejung (99%;
Siheung, Korea). Ampicillin, boric acid, and isopropyl-␤-d-
thiogalactopyranoside (IPTG) were purchased from USB (Cleveland,
OH, USA). All high-performance liquid chromatography (HPLC)
solvents were obtained from Duksan (HPLC grade; Ansan, Korea).
PCR primers were ordered from Bioneer (Daejeon, Korea).
2.5. Effects of solvent and detergent
The effect of solvent on the production of 9R-HODE was evalu-
ated using ethanol, methanol, isopropanol, butanol, acetone, ethyl
acetate, iso-octane, and hexane at the concentrations of 1% and 2%
(v/v). After each solvent was added to the reaction buffer, linoleic
acid was added. This mixture was sonicated for 2 min to disperse
and then was rapidly added to the solution of whole cells resus-
pended in the same reaction buffer on ice. The effect of methanol
concentration on the production of 9R-HODE was investigated by
varying its concentration from 0.5 to 5% (v/v). The effect of deter-
gent on the production of 9R-HODE was evaluated using Span 20,
Span 80, Tween 20, Tween 40, and Tween 80 at the concentrations
of 0.05 and 0.1% (w/v).
2.2. Microorganisms, media, and culture conditions
Nostoc sp. SAG 25.82, which was purchased from the culture col-
lection of algae (SAG, Goettingen, Germany), was cultivated at 26 ◦C
in BG-11 medium supplemented with 5 mM NaHCO3 as the inor-
ganic carbon source under 30 ␮mol photons m−2 s−1 as previously
described [26,28,29]. After 3 weeks of cultivation, cells were har-
vested by centrifugation at 3000 × g for 15 min and used as a cloning
source of 9R-LOX. E. coli ER 2566 (New England Biolabs, Hertford-
shire, UK) was used as the expression host of the 9R-LOX gene
and cultivated at 37 ◦C in Luria-Bertani (LB) medium containing
50 ␮g ml−1 ampicillin.
2.6. Effect of oxygen supplying method
Pure oxygen (O2) was supplied to a 500 ml baffled flask contain-
ing a working volume of 50 ml through an air flow meter (capacity,
1–500 ml min−1; Kojima, Kyoto, Japan) and double air regulators
connected to oxygen bombe via tubing. An air stone was attached
to the end of the tubing to achieve an even supply of bubbling oxy-
gen. Atmospheric air was supplied to the flask using an air pump
connected to the air flow meter. Oxygen or air supply was delivered
at a 50 ml min−1 l−1 (1 working volume/oxygen or air volume/min,
vvm) oxygenation or aeration rate without agitation or mixing.
Agitation was carried out in a shaking incubator (Vision Scientific,
Bucheon, Korea). Oxygenation or aeration with agitation took place
at a 1 vvm oxygenation or aeration rate and a 250 rpm agitation
speed. To investigate the effect of agitation speed on the produc-
tion of 9R-HODE, the agitation speed in a 500 ml baffled flask was
varied from 0 to 350 rpm in 50 rpm intervals.
2.3. Gene cloning and expression of 9R-LOX
Genomic DNA was prepared using a genomic DNA micro kit
(GeneAll, Seoul, Korea) according to the manufacturer manual. The
gene encoding 9R-LOX was amplified by PCR using the genomic
DNA of Nostoc sp. SAG 25.82 as a template. The primer sequences for
gene cloning were based on the reported DNA sequence of Nostoc
sp. PCC 7120 LOX (GenBank accession NC 003267; 17983-19293)
[30]. Forward (5ꢀ-AC CAT ATG CAG TAT TTG TAT GGA AGT AAG
GAT-3ꢀ) and reverse (5ꢀ-CA CTC GAGTAA ATG TTG ATA CTC ATC ATG
AG-3ꢀ) primers for the insertion pET-15b vector (Novagen, Darm-
stadt, Germany) were designed to introduce the Nde I and Xho I
restriction sites (underline), respectively. The amplified DNA frag-
ment obtained by PCR with ExTaq polymerase (Takara, Shiga, Japan)
was extracted using a gel extraction kit (Promega, Madison, WI,
USA). The PCR product was ligated into the Nde I and Xho I restric-
tion sites of pET-15b. The resulting plasmid was transformed into
2.7. Effects of cell and substrate concentrations
The cell concentration was varied from 10 to 60 g l−1 at a con-
stant linoleic acid concentration of 2 g l−1 to determine the optimal
cell concentration for the production of 9R-HODE using whole
recombinant cells expressing 9R-LOX. The substrate concentration
was varied from 5 to 45 g l−1 at a constant cell concentration of
40 g l−1 to determine the substrate concentration for the maximal
production of 9R-HODE. The reactions were performed for 30 min

58
K.-R. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 56–63
with a 1 vvm oxygenation rate and a 250 rpm agitation speed in a
500 ml baffled flask containing a working volume of 50 ml. Time
course reactions of the production of 9R-HODE from linoleic acid
using whole recombinant cells expressing 9R-LOX from Nostoc sp.
were conducted at 25 ◦C for 90 min in 50 mM Tris–HCl buffer (pH
7.5) containing 40 g l−1 cells, 15 g l−1 linoleic acid, and 2% (v/v)
methanol with a 1 vvm oxygenation rate and a 250 rpm agitation
speed in a 500 ml baffled flask containing a working volume of
50 ml.
3. Results and discussion
3.1. Properties of recombinant cells expressing LOX from Nostoc
sp.
The gene (1311 bp) encoding a LOX from Nostoc sp., with the
same sequence as that previously reported gene [30], was cloned
and expressed in the E. coli ER2566 host. The pET-15b plasmid har-
boring the LOX gene of Nostoc sp. was transformed into the host,
and the LOX protein was expressed in the recombinant cells by
IPTG induction. The expressed protein showed a single band on
SDS-PAGE with a molecular mass of approximately 48 kDa, which
is consistent with the calculated value of 48 kDa based on the 437
amino acids plus six histidine residues (data not shown). After 16 h
of induction, the activity of recombinant cells harboring LOX was
0.017 0.01 U mg−1 (dry cell weight).
2.8. Analytical methods
The cell mass was determined using a linear calibration curve
relating optical density at 600 nm to dry cell weight as previously
described [31]. Protein concentration was determined using the DC
protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with a
bovine serum albumin as a standard. Whole cell conversion was
determined as the amount of 9R-HODE from the initial linoleic
3.2. Identification of the product obtained from linoleic acid by
whole recombinant cells expressing LOX from Nostoc sp.
acid using 9R-HODE as a standard. The specific activity (U mg−1
)
of whole recombinant cells expressing 9R-LOX was defined as the
amount of cells required to produce 1 ␮mol of 9R-HODE per min
per unit amount of cells (mg dry cell weight) at 25 ◦C and pH 7.5
for 10 min. The protein expression of whole cells was examined
using sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The samples for SDS-PAGE analysis were prepared by
mixing whole cells and SDS buffer as a ratio of 1:4, and then dena-
tured for 10 min at 95 ◦C. After whole cell reactions, the reaction
broth was acidified with 3 M acetic acid to stop the reactions, and
the produced hydroperoxy fatty acid was reduced with NaBH4 or
SnCl2 on ice to convert the product into hydroxy fatty acid. The
reduced mixture was centrifuged at 12,000 × g at 4 ◦C for 5 min. The
supernatant was extracted with a 2-fold volume of ethyl acetate
twice, and dried using a nitrogen gas stream [26,32]. The dried
extract was dissolved in ethanol for HPLC analysis.
The reaction products of whole recombinant cells obtained
from linoleic acid were analyzed by HPLC using reverse-phase and
chiral-phase columns and LC-MS/MS (Fig. 1). Before reduction, two
distinct reaction products were detected to have the same reten-
tion times as the standards 9R-HODE and 9R-HPODE by HPLC using
a reverse-phase column (Fig. 1a). After reduction, a major reac-
tion product was observed with the same retention time as the
9R-HODE standard. The product obtained after reduction gave the
LC-MS/MS fragments as shown in the mass spectrum in Fig. 1b
[32,33]. The total molecular mass of the product was represented
by a peak at m/z 295 in full scan mode in LC-MS/MS. The molecu-
lar mass of HODE is 295. The peak at m/z 277 was formed by the
loss of H2O from the total molecular mass, and the peak at m/z
171 (−OOC(CH2)7C OH) was formed by cleavage of the adjacent
hydroxylated carbon of the C9 C10 bond. The small peak at m/z
251 was formed by the loss of CO2 from the product. The peaks
at m/z 295, m/z 277, and m/z 251 indicated that the product was
HODE, and the peak at m/z 171 also indicated that the product
was hydroxylated at the C9 position. LC-MS/MS peaks of the 9R-
HODE standard showed the same fragmentation pattern as the
reaction product of whole recombinant cells obtained from linoleic
acid (data not shown). The chirality of the 9-HODE product was
confirmed by HPLC using a chiral-phase column with the 9R-, 9S-
, and 13S-HODE standards. The major product 9R-HODE and the
minor product 13S-HODE were detected using the same retention
times of their standards (Fig. 1c). Therefore, the Nostoc sp. LOX was
a 9R-LOX, and whole recombinant cells harboring Nostoc 9R-LOX
converts linoleic acid to 9R-HODE.
2.9. Liquid chromatography-mass spectrometry
The formations of 9R- and 13S-HODE and 9R-HPODE were ana-
lyzed using an HPLC system (Agilent 1260, Palo Alto, CA, USA)
coupled to a UV detector at the absorbance at 234 nm with a reverse
phase Nucleosil C18 column (150 mm × 2.1 mm, 5 ␮m particle size;
Phenomenex, Torrance, CA, USA) [28,32]. The column was eluted at
25 ◦C with a gradient of solvent A (acetonitrile/water/acetic acid;
50:50:0.1, v/v/v) and solvent B (acetonitrile/acetic acid; 100:0.1,
v/v). The chirality of the product was determined using HPLC with
a chiral phase Chiralcel OD-H column (150 mm × 2.1 mm, 5 ␮m
particle size; Daicel, Tokyo, Japan). The column was eluted at a
flow rate of 0.15 ml min−1 with a solvent system of n-hexane/2-
propanol/acetic acid (100:5:0.1, v/v/v).
3.3. Effects of pH, temperature, and stability on the production of
9R-HODE by whole recombinant cells expressing 9R-LOX from
Nostoc sp.
Liquid chromatography-mass spectrometry/mass spectrometry
(LC-MS/MS) analysis of HFAs was performed using a ThermoFinni-
gan LCQ Deca XP plus ion trap mass spectrometer (Thermo
˚
Scientific, Pittsburg, PA, USA) with a Synergi fusion-RP 80 A column
The effects of pH and temperature on the production of 9R-
HODE from linoleic acid as a substrate by whole recombinant cells
expressing 9R-LOX were determined by using reverse phase-HPLC.
The optimum pH of the whole cells was 7.5, and the production of
9R-HODE was rapidly declined at lower and higher pH. The activ-
ity in sodium phosphate buffer was approximately 80% of that
in Tris–HCl buffer (Fig. 2a). The optimal pH of purified LOX from
Anabaena sp. PCC 7120 expressed in Bacillus subtilis was 6.0 [34].
The activity of purified LOX from Nostoc punctiforme PCC 73102
for the production of 13S-HODE using linoleic acid as a substrate
showed a broad optimum pH in the range of 4.5–8.0 [29]. There-
fore, the optimum pH of recombinant cells expressing 9R-LOX from
Nostoc sp. was different from those of other LOXs.
(100 mm × 30 mm, 4 ␮m particle size; Phenomenex) at National
Instrumentation Center for Environmental Management (NICEM,
Seoul, Korea) using an electrospray ionization (ESI) interface. The
flow rate was 250 ␮l min−1 and a gradient of 0.1% formic acid and
acetonitrile was used. The instrument consisted of an LC pump,
an auto sampler, and a photodiode array detector. The following
operation parameters were used: capillary temperature, 275 ◦C;
ion source voltage, 5 kV; nebulizer gas, 40 psi nitrogen; capillary
voltage, 46 V in positive mode and 15 V in negative ionization
mode; average scan time, 0.01 min; average time to change polarity,
0.02 min; and collision energy (CE), approximately 35% abundance
of the precursor ion.

K.-R. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 56–63
59
Fig. 1. Product analysis of whole recombinant cells harboring LOX from Nostoc sp. obtained in the presence of the linoleic acid substrate. (a) HPLC chromatogram using
a reverse-phase column. Reaction products of Nostoc sp. LOX, upper; the 9R-HODE and 9R-HPODE standards, middle; and the linoleic acid substrate, lower. (b) LC-MS/MS
chromatogram. The inset shows formation of the major fragments. (c) HPLC chromatogram using a chiral-phase column. Reaction products of Nostoc sp. LOX, upper; and the
9R- (2), 9S- (3), and 13S-HODE (1) standards, lower.
The optimum temperature of whole recombinant E. coli cells
containing Nostoc sp. 9R-LOX was 25 ◦C, and its activity at 30 ◦C was
approximately 95% of maximum activity at 25 ◦C (Fig. 2b). The opti-
mum reported temperatures of LOXs were 45 ◦C for Anabaena sp.
PCC 7120 LOX expressed in B. subtilis, [34] and 25 ◦C for P. aeruginosa
13S-LOX expressed in E. coli [35]. The thermal stability of whole
recombinant cells expressing 9R-LOX was assayed after incuba-
tion for 3.5 h. The highest activity was obtained at 25 ◦C, and the
enzyme activity at this temperature was not decreased after 3.5 h.
Above 25 ◦C, the activity of recombinant cells expressing 9R-LOX
decreased as the temperature increased and then the activity was
approximately 50% of that at 40 ◦C (Fig. 2c).
was examined by varying its concentration from 1 to 5% (v/v).
Methanol at 2% was determined to be the optimum concentra-
tion, resulting in a 1.2-fold increase in the production of 9R-HODE,
compared to that without methanol (Fig. 3b). Methanol supple-
mentation may be helpful to solubilize the substrate linoleic acid
and to enhance contact between substrate and enzyme, resulting
in the increase of conversion rate. When methanol within 5% (v/v)
was supplemented, no cell lysis was observed. However, the addi-
tion of detergent, including Span 20, Span 80, Tween 20, Tween
40, or Tween 80, at a concentration of 0.05 or 0.1% (w/v) did not
increase the production of 9R-HODE (data not shown).
3.5. Effect of oxygen supply method on the production of
9R-HODE by whole recombinant cells expressing 9R-LOX
3.4. Effects of solvent and detergent on the production of
9R-HODE by whole recombinant cells expressing 9R-LOX from
Nostoc sp.
The effect of oxygen supply method on the production of
9R-HODE by whole recombinant cells expressing 9R-LOX was
investigated without oxygenation, aeration, and agitation and with
aeration (1 vvm), oxygenation (1 vvm), agitation (250 rpm), aera-
tion (1 vvm) plus agitation (250 rpm), and oxygenation (1 vvm) plus
agitation (250 rpm) in a 500 ml baffled flask (Fig. 4a). The produc-
tion of 9R-HODE using all kinds of oxygen supply methods was
over 5-fold higher than that when no oxygen supply was used.
Among the oxygen supply methods tested, oxygen supplemented
agitation method showed the highest production of 9R-HODE. The
The effect of solvent on the production of 9R-HODE was investi-
gated by 10 g l−1 whole recombinant cells expressing 9R-LOX with
2 g l−1 linoleic acid at 25 ◦C and pH 7.5 supplemented with 1 or
2% (v/v) solvent. The production of 9R-HODE from linoleic acid by
whole recombinant cells expressing 9R-LOX was the highest using
methanol among all of the solvents tested (Fig. 3a). Ethanol also
showed similar enhancing effect for the production of 9R-HODE.
The effect of methanol concentration on the production of 9R-HODE

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K.-R. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 56–63
(a)
(b)
120
120
100
80
60
40
20
0
100
80
60
40
20
0
6.5
7.0
7.5
8.0
8.5
10
15
20
25
30
35
40
45
Temperature (oC)
pH
(c)
120
100
80
60
40
20
0
5
10
15
20
25
30
35
40
45
Temperature (oC)
Fig. 2. Effects of pH (a), temperature (b), and stability (c) on the production of 9R-HODE by whole recombinant cells expressing 9R-LOX from Nostoc sp. The reactions for
pH experiments were performed at 25 ◦C for 20 min using 100 mM sodium phosphate buffer (ꢁ, pH 6.5–8.0) and 50 mM Tris–HCl buffer (᭹, pH 7.0–8.5) containing 10 g l−1
cells and 2 g l−1 linoleic acid. The stability of whole recombinant cells was measured after incubation for 3.5 h. At 100% relative activity, 0.7 and 0.94 g l−1 of 9R-HODE was
produced in the pH and temperature experiments, respectively.
effects of oxygenation with and without agitation on the produc-
tion of 9R-HODE were slightly stronger than those of aeration with
and without agitation, respectively. The conversion rate of soybean
LOX by oxygenation (O2 supply) was 1.4-fold higher than that by
aeration (atmosphere air supply) [36]. Interestingly, agitation was
more effective for the production of 9R-HODE than aeration or oxy-
genation because the used baffled flask may have enhanced the
mixing and reduced the bubble sizes. At agitation speeds below
250 rpm, the production of 9R-HODE from linoleic acid increased
with increasing agitation speed, however, the production plateaued
above 250 rpm (Fig. 4b).
13-HPODE from linoleic acid, increases the 13/9-HPODE ratio
[37].
3.6. Effects of cell and substrate concentrations on the production
of 9R-HODE by whole recombinant cells expressing 9R-LOX from
Nostoc sp.
The optimal concentration of whole recombinant cells express-
ing 9R-LOX to produce 9R-HODE was investigated by varying the
Table 1
The effect of oxygen supply method on the 9R-/13S-HODE ratio
was investigated (Table 1). Oxygen supplemented agitation showed
the highest 9R/13S-HODE ratio (96:4) of the products, whereas no
oxygen supply method showed the lowest ratio (87:13). Moreover,
oxygen is a crucial co-substrate for the insertion of molecular oxy-
gen into an unsaturated fatty acid. Therefore, oxygen supplemented
agitation using whole recombinant cells harboring 9R-LOX was the
most effective method to obtain a high 9R/13S-HODE ratio and to
enhance the production of 9R-HODE. However, the increase of oxy-
gen concentration in the reaction of soybean LOX, which produces
Effect of oxygen supply method on the 9R-/13S-HODE ratio.
Air source
9R-HODE (%)
13S-HODE (%)
9R/13S Ratio
Oxygenation (1 vvm)
Aeration (1 vvm)
95.4
91.3
96.2
4.6
8.7
3.8
20.7
10.5
25.3
Oxygenation (1 vvm) with
agitation (250 rpm)
Aeration (1 vvm) with
agitation (250 rpm)
Agitation (250 rpm)
No aeration and agitation
93.7
6.3
14.9
93.8
87.3
6.2
12.7
15.1
6.3

K.-R. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 56–63
61
(a)
(a)
1600
140
120
100
80
1400
1200
1000
800
600
400
200
0
60
40
20
0
O2+
Agitation
O2
Air +
Agitation
Control Agitation Air
te
eta
so-oc
ne
ne
ta
a
one
t
ontrol
hanol
I
t
a
ex
C
Ethanol
Me
utanol
ac
ce
H
B
A
yl
sopropanol
I
Eth
Air supplying methods
(b)
(b)
140
120
100
80
120
100
80
60
40
20
0
0
0
1
2
3
4
5
6
0
100
200
300
400
Methanol (%, v/v)
Agitation speed (rpm)
Fig. 3. Effects of solvent (a) and methanol concentration (b) on the production of 9R-
HODE by whole recombinant cells expressing 9R-LOX from Nostoc sp. The reactions
were performed at 25 ◦C for 20 min in 50 mM Tris–HCl buffer (pH 7.5) containing
10 g l−1 cells, 2 g l−1 linoleic acid, and 1 (ꢀ) or 2% (v/v) (ꢀ) solvent. A control reaction
was performed without solvent. At 100% relative activity, 1.1 g l−1 of 9R-HODE was
produced.
Fig. 4. Effects of oxygen supply method (a) and agitation speed (b) on the produc-
tion of 9R-HODE by whole recombinant cells expressing 9R-LOX from Nostoc sp.
The reactions were performed at 25 ◦C for 20 min under different oxygen supply
conditions in 50 mM Tris–HCl buffer (pH 7.5) containing 10 g l−1 cells, 2 g l−1 linoleic
acid, and 2% (v/v) methanol. The agitation speed for oxygen supply was 250 rpm and
the aeration and oxygenation rates were 1 vvm in a 500 ml baffled flask containing
a working volume of 50 ml. The control reaction was the production of 9R-HODE
without oxygenation, aeration, and agitation. At 100% relative activity for blank,
0.1 g l−1 of 9R-HODE was produced.
cell concentration from 10 to 60 g l−1 with 2 g l−1 linoleic acid
for 40 min. Below a cell concentration of 40 g l−1, the production
of 9R-HODE and conversion yield increased with increasing cell
concentration, however, they decreased above 40 g l−1 (Fig. 5a).
Therefore, the optimal cell concentration was determined to be
40 g l−1. The production of 9R-HODE from linoleic acid was exam-
ined by varying the substrate concentrations from 5 to 45 g l−1
while holding the cell concentration constant at 40 g l−1 cells for
40 min. Below a substrate concentration of 30 g l−1, increases in
the substrate concentration led to increase in the production of
9R-HODE, however, these increases did not occur above 30 g l−1
linoleic acid (Fig. 5b). Above 15 g l−1 linoleic acid, the conversion
yield decreased with increasing substrate concentration and then
was 39% (w/w) at 45 g l−1 linoleic acid. When the substrate concen-
tration was greater than 15 g l−1, the conversion yield decreased
significantly because the high viscosity of the reaction solution
became high resistance to mass transfer. Thus, the substrate con-
3.7. Production of 9R-HODE by whole recombinant cells
expressing 9R-LOX from Nostoc sp. under the optimized conditions
The optimal conditions of whole recombinant E. coli cells
expressing 9R-LOX from Nostoc sp. to produce 9R-HODE were pH
7.5, 25 ◦C, 40 g l−1 cells, 15 g l−1 linoleic acid, 2% (v/v) methanol, a
1 vvm oxygenation rate, and a 250 rpm agitation speed in a 500 ml
baffled flask containing a working volume of 50 ml. Under these
optimized conditions, whole recombinant cells expressing 9R-LOX
produced 14.3 g l−1 9R-HODE for 1 h with a conversion yield of 95%
(w/w), volumetric productivity of 14.3 g l−1 h−1, specific productiv-
ity 0.95 g l−1 h−1, and 9R/13S-HODE ratio of 96:4 (Fig. 6).
The mechanism of stereo-selective oxygenation of LOX using
linoleic acid as a substrate occurs by the stereo-specific hydro-
gen abstraction at the C11 position of linoleic acid, and then
regio- and stereo-specific oxygen insertion takes place in antarafa-
cial manner at either the C9 or C13 position to produce 9- or
centration was determined to be 15 g l−1
.

62
K.-R. Kim et al. / Journal of Molecular Catalysis B: Enzymatic 104 (2014) 56–63
13-hydroperoxide, respectively [2]. Plant LOXs catalyze the inser-
120
100
80
60
40
20
0
(a)
tion of oxygen with S stereo-specificity to produce 9S-specific
fatty acids, whereas cyanobacteria produce 9R-hydroxy fatty acids
[26,27]. The determinant residues for S-LOX and R-LOX have con-
served as valine and alanine residues [38,39], respectively. The
9R-LOX from Nostoc sp. SAG 25.82 used in the present study has
a conserved alanine residue, indicating that the LOX is confirmed
as an R-LOX.
HPFAs have been produced using either crude extracts or partial
purified enzymes. However, these protein sources contain other
enzymes and unknown proteins that result in the utilization of
PUFA substrates or hydroperoxide products, leading to reduced
activity [40]. Alternatively, purified recombinant LOX has been sug-
gested to increase the catalysis of LOX with PUFA substrates [34,40].
However, the conversion using purified enzyme is laborious and
requires the need for purification steps, such as cell lysis, precip-
itation, and dialysis. Thus, this is the first study in which whole
recombinant cells have been used in the stereo-selective produc-
tion of 9R-HODE. No LOX is present in E. coli and there are no
by-products or unwanted reactions, thus, whole cell conversion is
less laborious and eliminates the need for purification steps [41].
0
10
20
30
40
50
60
70
Cell concentration (g l-1)
(b)
120
100
80
60
40
20
0
25
20
15
10
5
4. Conclusion
We successfully achieved the stereo-specific production of R-
HFA using whole recombinant E. coli cells expressing R-specific
LOX from Nostoc sp. Oxygen supplemented agitation was the most
effective method not only to obtain a high 9R/13S-HODE ratio, but
also to enhance the production of 9R-HODE. The pH, temperature,
cell and substrate concentrations, and solvent to produce 9R-HODE
from linoleic acid using oxygen supplemented agitation were opti-
mized. Under these optimized conditions, whole recombinant cells
expressing R-LOX produced 14.3 g l−1 9R-HODE for 1 h, with a con-
version yield of 95% (w/w), and an R/S ratio of 96:4. Therefore,
our approach using whole cells expressing R-specific LOX is a cost-
effective method for the stereo-specific production of HFA.
0
0
10
20
30
40
50
-1
Linoleic acid (gl
)
Fig. 5. Effects of cell (a) and substrate (b) concentrations on the production of 9R-
HODE by whole recombinant cells expressing 9R-LOX from Nostoc sp. The agitation
speed for oxygen supply was 250 rpm and the oxygenation rate was 1 vvm in a
500 ml baffled flask containing a working volume of 50 ml. Production of 9R-HODE
(᭹) and conversion yield of 9R-HODE from linoleic acid (ꢁ). At 100% relative activity,
1.9 g l−1 of 9R-HODE was produced from 2 g l−1 linoleic acid.
Acknowledgments
This study was supported by grants from the Bio-industry
Technology Development Program, Ministry for Food, Agriculture,
Forestry and Fisheries (No. 112002-3) and the Korea Healthcare
Technology R&D Project, Ministry for Health & Welfare, Republic of
Korea (No. 2012-009).
18
15
12
9
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