One-Step Bioconversion of Fatty Acids into C8-C9 Volatile Aroma Compounds by a Multifunctional Lipoxygenase Cloned from Pyropia haitanensis

Article abstract of DOI:10.1021/acs.jafc.7b05341

The multifunctional lipoxygenase PhLOX cloned from Pyropia haitanensis was expressed in Escherichia coli with 24.4 mg·L^-1 yield. PhLOX could catalyze the one-step bioconversion of C18-C22 fatty acids into C8-C9 volatile organic compounds (VOCs), displaying higher catalytic efficiency for eicosenoic and docosenoic acids than for octadecenoic acids. C20:5 was the most suitable substrate among the tested fatty acids. The C8-C9 VOCs were generated in good yields from fatty acids, e.g., 2E-nonenal from C20:4, and 2E,6Z-nonadienal from C20:5. Hydrolyzed oils were also tested as substrates. The reactions mainly generated 2E,4E-pentadienal, 2E-octenal, and 2E,4E-octadienal from hydrolyzed sunflower seed oil, corn oil, and fish oil, respectively. PhLOX showed good stability after storage at 4 °C for 2 weeks and broad tolerance to pH and temperature. These desirable properties of PhLOX make it a promising novel biocatalyst for the industrial production of volatile aroma compounds.

Full text of DOI:10.1021/acs.jafc.7b05341

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Article
One-step bioconversion of fatty acids into C8–C9 volatile aroma compounds
by a multifunctional lipoxygenase cloned from Pyropia haitanensis
Zhu-Jun Zhu, Hai-Min Chen, Juanjuan Chen, Rui Yang, and Xiao-Jun Yan
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05341 • Publication Date (Web): 12 Jan 2018
Downloaded from http://pubs.acs.org on January 12, 2018
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One-step bioconversion of fatty acids into C8–C9 volatile aroma compounds by a
multifunctional lipoxygenase cloned from Pyropia haitanensis
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ZhuꢀJun Zhu1,2, HaiꢀMin Chen1,*, JuanꢀJuan Chen1, Rui Yang1, XiaoꢀJun Yan1,*
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1 Key Laboratory of Marine Biotechnology of Zhejiang Province, Ningbo University,
Ningbo, Zhejiang, 315211, China.
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2 Ningbo Institute of Oceanography, Ningbo, Zhejiang, 315832, China.
* Corresponding authors
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ABSTRACT: The multifunctional lipoxygenase PhLOX cloned from Pyropia
haitanensis was expressed in E. coli with 24.4 mg·L−1 yield. PhLOX could catalyze
the oneꢀstep bioconversion of C18–C22 fatty acids into C8–C9 volatile organic
compounds (VOCs), displaying higher catalytic efficiency for eicosenoic and
docosenoic acids than for octadecenoic acids. C20:5 was the most suitable substrate
among the tested fatty acids. The C8–C9 VOCs were generated in good yields from
fatty acids, e.g., 2Eꢀnonenal from C20:4, and 2E,6Zꢀnonadienal from C20:5.
Hydrolyzed oils were also tested as substrates. The reactions mainly generated
2E,4Eꢀpentadienal, 2Eꢀoctenal, and 2E,4Eꢀoctadienal from hydrolyzed sunflower seed
oil, corn oil, and fish oil, respectively. PhLOX showed good stability after storage at
4 °C for two weeks and broad tolerance to pH and temperature. These desirable
properties of PhLOX make it a promising novel biocatalyst for the industrial
production of volatile aroma compounds.
KEYWORDS: Pyropia haitanensis; lipoxygenase; hydroperoxide lyase; volatile
organic compounds; aroma
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INTRODUCTION
Among the many flavor compounds commonly used in food industry, certain
C6–C9 aldehydes and alcohols are particularly remarkable because they can
reintroduce the "fresh green" flavor of fruits and vegetables that is lost during
processing.1,2 These compounds include 2Eꢀ/3Zꢀhexenal and hexenol, 1ꢀ/3ꢀoctanol,
2Zꢀoctenꢀ1ꢀol, 1ꢀoctenꢀ3ꢀol, 1ꢀoctenꢀ3ꢀone, 2Eꢀnonenal, as well as 2E,6Zꢀnonadienal.
For example, 1ꢀoctenꢀ3ꢀol can give a unique and subtle flavor of fresh mushrooms,3
and 2Eꢀnonenal and 2E,6Zꢀnonadienal can provide a fresh flavor that resembles
cucumber.4
Flavor compounds used to be extracted directly from biomaterials,5 but the
increasing market demand required the industry to exploit biocatalysts to synthesize
flavor compounds.6 Studies have shown that in enzymatic catalysis, volatile organic
compounds (VOCs) can be produced from polyunsaturated fatty acids (PUFAs) in two
steps. In the first step, the PUFAs are peroxided by lipoxygenase (LOX). Secondly,
the hydroperoxides are cleaved by hydroperoxide lyase (HPL) to produce short chain
aldehydes. The aldehydes may be additionally reduced to the corresponding alcohols
by alcohol dehydrogenase (ADH).7
In industry, the biocatalyst for the synthesis of VOCs can be obtained in two
ways. Traditionally, a homogenate from plant materials that contains the enzymatic
mixture can be used.8ꢀ10 For example, 1ꢀoctenꢀ3ꢀol could be produced from linoleic
acid by using the homogenate from the mushroom Agaricus bisporus as a biocatalyst,
which contained the LOX and the associated HPL.11 It was also reported that the LOX
in the homogenate of the mushroom Psalliota bispora could catalytically convert
linoleic acid into its 10ꢀhydroperoxide, which was then cleaved at the ωꢀ8 position to
produce 1ꢀoctenꢀ3ꢀol by the HPL in the homogenate.12 Nevertheless, the activities of
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enzymes in plant homogenates are affected by many factors, e.g., the varieties and
sources of the plant materials, the quality of homogenate, etc. Moreover, coexisting
enzymes in the homogenates may result in undesirable byproducts or impede the
conversion of substrates.
Alternatively, the biosynthesis of VOCs may make use of recombinant LOX and
HPL enzymes cloned from different plants.13ꢀ15 For example, the soybean LOX and
watermelon HPL genes were cloned and coꢀoverexpressed in the yeast
Saccharomyces cerevisiae, and it was found that the recombinant cells could convert
linolenic acid to 2Eꢀhexenal and 3Zꢀhexenal.16 Although pure recombinant LOXꢀHPL
can increase the catalytic efficiency, the two enzymes need to finish disparate reaction
steps at the same time, which can make it difficult to manage the optimal reaction
condition, especially since HPL can be easily inactivated in vitro.17 As a result,
researchers have been actively exploring novel and efficient biocatalysts to prepare
aroma compounds.
We have previously cloned a lipoxygenase gene from the red alga Pyropia
haitanensis.18 The obtained enzyme, denoted as PhLOX, was found to be a unique
multifunctional lipoxygenase that possessed exceptionally high HPL, LOX, and allene
oxide synthase (AOS) activities within only one catalytic domain of the protein.
Hence, PhLOX may be a promising enzyme for the oneꢀstep biosynthesis of aroma
compounds. In this work, we determined the enzymatic activities of PhLOX for
different PUFAs and hydrolyzed oils. We also measured the kinetic parameters, the
optimal enzymatic conditions, and the enzymatic stability of PhLOX.
MATERIALS AND METHODS
Expression and Purification of PhLOX. The recombinant strain, E. coli
PhLOXꢀpET28aꢀBL21 (DE3), was provided by the Key Laboratory of Applied
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Marine Biotechnology of Zhejiang Province. The PhLOX gene was cloned from P.
haitanensis HML thallus (GenBank Accession No. AFQ59981). Its cloning and
recombination has been described in our previous work.18 Five hundred milliliters of
LuriaꢀBertani medium was incubated with 5 mL of the overnight culture of the
recombinant strain and incubated at 37 °C until the cell density had increased to A600
0.6 to 0.8. The expression of PhLOX was then induced by 0.1 mM IPTG at 16 °C for
20 h. The cells were harvested by centrifuging for 10 min at 8,000 rpm (10,800 g) and
4 °C, reꢀsuspended by adding 20 mM TrisꢀHCl buffer A (200 mL, pH = 8.0, 200 mM
NaCl, 0.1% Tween 20, 5% glycerin), lysed by ultrasonication, and centrifuged for 10
min at 12,000 rpm (17,226 g) and 4 °C. The supernatant of cell lysis was purified by
affinity chromatography using NiꢀAgarose 6× HisꢀTagged Protein Purification Kit
(CWBIO, China). Protein concentration was measured with the DCTM Protein Assay
Kit (BioRad, USA). Protein purity was evaluated by 12% SDSꢀPAGE analysis.
Measurements of PhLOX Activity and Kinetic Parameters. The fatty acid
substrates, which included linoleic acid (LA), αꢀlinolenic acid (αꢀALA), arachidonic
acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were
purchased from the Cayman Chemical, USA. In a typical test, 1 ꢁM PhLOX (about
0.1 mg·mL−1) was incubated with 100 ꢁM substrate in 20 mM TrisꢀHCl buffer B (1
mL, pH = 8.0) containing 200 mM NaCl at 20 °C for different times (6 s, 12 s, 0.5, 1,
5, 10, 15, 30, 60 min). In the control group, PhLOX was inactivated by boiling for 5
min. The reaction was quenched by acidification (pH < 2) with 4 M HCl, added with
1 mL ethyl acetate and shaken at 4 °C for 20 min. The mixture was then centrifuged
for 10 min at 12,000 rpm (13,400 g) and 4 °C. The organic phase was washed with 1
mL water, dried with a stream of nitrogen, metered volume in 0.5 mL methanol, and
then analyzed by HPLCꢀMS to determine the composition of fatty acids. All assays
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were run in triplicate.
The activity of PhLOX was determined by monitoring the consumption of fatty
acid substrates, and one unit of PhLOX activity corresponded to 1 µM fatty acid
consumed in one minute by 1 mg PhLOX. External standards were used to quantify
the substrates consumed by PhLOX, and the calibration curves were built with the
five fatty acids mentioned above.
Kinetic parameters were determined at varying substrate concentrations (50, 100,
150, 200, 300, 400, 500, 650 and 800 µM). According to the reaction curve of each
fatty acid, the reaction period for LA, αꢀALA, ARA, EPA, and DHA was 3 min, 3 min,
6 s, 6 s, and 12 s, respectively. Data were fitted to the Michaelis–Menten model to
determine the values of the Michaelis constant KM and the maximum rate Vm, and kcat
was calculated from Equation (1):
kcat = Vm/[E]
(1)
where [E] is the concentration of the enzyme (in micromolar).
Citric acid/sodium citrate buffer (20 mM, pH = 2.5–5.0), sodium phosphate
buffer (20 mM, pH = 6.0–7.0), TrisꢀHCl buffer (20 mM, pH = 8.0–10.0) and sodium
bicarbonate/sodium hydroxide buffer (20 mM, pH = 11.0) were tested as the buffer
medium to determine the optimal pH for the reaction. The enzymatic activity was also
measured at 0, 4, 10, 20, 30, 40, 50, 55 and 60 °C to determine the optimal
temperature. The reaction period for LA, αꢀALA, ARA, EPA, and DHA in incubation
with PhLOX or inactive PhLOX (as the control) were 3 min, 3 min, 6 s, 6 s, and 12 s,
respectively, too. Additionally, a mixture of five PUFAs in 1 mL inactive PhLOX was
made and extracted immediately to detect any nonꢀenzyme related substrate depletion
by comparing to the fatty acid contents after incubation in the control.
HPLC-MS analysis. The HPLCꢀMS assay was performed on a Finnigan
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Surveyor with a TSQ Quantum Access system (Thermo Fisher Scientific Inc.,
Pittsburgh, PA, USA) using a Hypersil GOLD C18 column (2.1 mm × 100 mm, 3 ꢁm,
Thermo Fisher Scientific Inc.) under the following conditions: injection volume, 10
ꢁL; column temperature, 30 °C; flow rate, 0.2 mL·min−1. The binary eluent consisted
of (A) acetonitrile and (B) 0.2% acetic acid. The gradient elution was programmed to
increase from 30% A to 100% A over 40 min.
Mass spectrometry was performed on a triple quadrupole mass spectrometer
equipped with electrospray ionization (ESI) and operated in negative ionization mode.
Samples were scanned over m/z = 100–600. The ionization conditions were: sheath
gas pressure (N2) at a flow rate of 25 L·min−1, auxiliary gas pressure (N2) at a flow
rate of 5 L·min−1, spray voltage at 2.5 kV, vaporizer temperature at 300 °C, and
capillary temperature at 350 °C. Argon was introduced into the trap at an estimated
pressure of 6 × 10−6 mbar to improve trapping efficiency and to act as the collision
gas. The collision gas pressure was 1.5 mTorr.
Preparation and Component Analysis of Hydrolyzed Oils. Sunflower seed oil,
corn oil, and fish oil were purchased from a local supermarket. These oils (0.5 g) were
hydrolyzed in MeOH/H2O (4:1 v/v, 2 mL) containing 6% KOH under N2 atmosphere
at 60 °C for 2 h. The fatty acid products were collected by firstly cooling the reaction
mixture to room temperature, then acidification (pH < 2) with 4 M HCl, and finally
extraction with hexane/chloroform (4:1 v/v, 1 mL) for three times. The collected
hydrolyzed oils were dried with a stream of nitrogen, dissolved in 1 mL methanol and
stored at −20 °C.
To analyze the components of hydrolyzed oils after incubation with PhLOX, a
mixture containing NaCl (200 mM), PhLOX (1 ꢁM), and hydrolyzed oil (2 ꢁL) in 20
mM TrisꢀHCl buffer B (2 mL, pH = 8.0) was maintained at 20 °C for 1 h. A boiled
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PhLOX and otherwise identical mixture was set up as the control group, and in both
cases C19:0 (15 ꢁg) was added as an internal standard (IS). After incubation, the
reaction mixture was quenched by acidification (pH < 2) with 4 M HCl, added with
2×1 mL ethyl acetate and shaken at 4 °C for 20 min. The extracts were collected and
dried with a stream of N2, then heated in 14% BF3·CH3OH (500 ꢁL) at 60 °C for 1 h,
and finally extracted successively with hexane/chloroform (4:1 v/v, 0.2 mL, twice)
and hexane (0.2 mL, once). The combined extracts were mixed with MilliꢀQ water (1
mL) and centrifuged for 15 min at 12,000 rpm (13,400 g) and 4 °C. The upper layer
was separated and dehydrated to give the incubated sample. The sample was then
dried with a stream of N2, redissolved in hexane (0.2 mL), and analyzed on a QP2010
GCꢀMS (Shimadzu, Kyoto, Japan). Additionally, 2 ꢁL hydrolyzed oil was added into
2 mL boiled PhLOX, and then extracted and derived immediately by following the
process above to analyze the fatty acid composition of hydrolyzed oil before
incubation.
The GCꢀMS assay was carried out using a SPBꢀ50 fused silica capillary column
(30 m × 0.25 mm × 0.25 ꢁm; Supelco Inc., Bellefonte, PA, USA) under the following
conditions: injector temperature, 250 °C; carrier gas, He; constant flow, flow rate,
0.81 mL·min−1; preꢀcolumn pressure, 73.0 kPa; injection volume, 1 ꢁL; split ratio,
50:1. The oven temperature was programmed as follows: hold at 150 °C for 3.5 min
after injection, raise to 200 °C at 20 °C·min−1 and hold for 5 min, and raise to 280 °C
at 5 °C·min−1 and hold for 30 min. The mass spectrometer was operated in the
electron impact mode under the following conditions: electron energy, 70 eV; ion
source temperature, 200 °C; interface temperature, 250 °C. The mass spectrometer
scanned over m/z = 50–600. The polyunsaturated fatty acid components were
expressed in micromolar, and their consumption rate was calculated from Equation
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(2):
RFA (%) = ([FA]0 − [FA]t)/[FA]0 × 100%
(2)
where RFA denotes the consumption rate of different fatty acids in the hydrolyzed oils,
[FA]0 is the concentration of the fatty acid after incubation with inactive PhLOX, and
[FA]t is the concentration of the fatty acid after incubation with PhLOX.
Production of Volatile Compounds. The substrates for the production of aroma
compounds with PhLOX included the five pure fatty acids (LA, αꢀALA, ARA, EPA,
and DHA) and the three hydrolyzed oil samples. In a typical reaction, a mixture of
NaCl (200 mM), PhLOX (1 ꢁM), and the pure fatty acid (100 ꢁM) or hydrolyzed oil
(2 ꢁL) was maintained in 20 mM TrisꢀHCl buffer B (2 mL final volume, pH = 8.0) at
20 °C for 1 h in a closed tube. The boiled PhLOX was used in the control group. The
reaction was stopped by acidification (pH < 2) with 4 M HCl, and vanillin (200 ꢁg)
was added as an internal standard (IS).
The products were concentrated on a solid phase micro extraction (SPME) fiber
assembly polydimethylsiloxane/carboxen/divinylbenzene (Supelco Inc., Bellefonte,
PA, USA). 19, 20 The extraction was performed in headspace mode at 40 °C for 50 min
with magnetic stirring. After extraction, the SPME device was introduced into the
splitless injector of GCꢀMS and maintained at 210 °C for 5 min, and the products
were analyzed on the QP2010 GCꢀMS fitted with a VOCOL column (60 m × 0.32 mm,
1.8 ꢁm film thickness) (Supelco Inc., Bellefonte, PA, USA) under the following
conditions: carrier gas, He; constant flow, flow rate, 1.99 mL·min−1; preꢀcolumn
pressure, 83.5 kPa. The oven temperature was programmed as follows: hold at 35 °C
for 3 min, raise to 40 °C at 3 °C·min−1 and hold for 1 min, raise to 100 °C at
5 °C·min−1, and finally raise to 210 °C at 10 °C·min−1 and hold for 30 min. Another
column (Agilent HPꢀ5MS, 30 m × 0.25 mm × 0.25 ꢁm) was also used. The oven
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temperature was programmed as follows: hold at 35 °C for 3 min, raise to 50 °C at
3 °C·min−1 and hold for 1 min, raise to 180 °C at 6 °C·min−1 and hold for 2 min, and
finally raise to 270 °C at 15 °C·min−1 and hold for 7 min. The mass spectrometer was
operated in the electron impact mode under the following conditions: electron energy,
70 eV; ion source temperature, 200 °C; interface temperature, 210 °C. The mass
spectrometer scanned over m/z = 45–1000.
Commercial volatile aroma compounds were used as standard compounds. They
were mixed in 2 mL buffer B and then handled by the same procedure of SPME and
GCꢀMS above. Both retention time and mass spectra of analytes were compared with
those of the standard compounds. The mass spectra of analytes were also compared
with the standard spectra recorded in the NIST 147, the NIST 27 and the WILEY 7
Spectrometry Libraries. The quantification of analytes was calculated from Equation
(3) and expressed in micromolar.
Cp=
[
(mIS×Ap)/(f×AIS)]/(Mp×Vr)
(3)
where Cp denotes the concentration of the volatile product, mIS is the mass of the
internal standard (200 ꢁg), Ap is the peak area of the volatile product, f is the relative
weight correction factor calculated by the peak area ratio of the standard compound
(200 ꢁg) to the internal standard (200 ꢁg), AIS is the peak area of the internal standard
(200 ꢁg), Mp is the molecular weight of the volatile product and Vr is the final volume
of the enzymatic reaction (2 mL).
The yield of the volatile products was calculated from Equation (4):
Yp (%) = (Cp/[S]) × 100%
(4)
where Yp denotes the yield, Cp is the concentration of the volatile product, and [S] is
the initial substrate concentration (in micromolar).
The substrate conversion ratio (RS) was expressed in the percentage (%) of the
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consumed substrate in the total substrate.
Volatile compounds in three hydrolyzed oils were semiꢀquantitatively determined
by using vanillin as an internal standard and the contents of the volatiles were
calculated from the GC peak areas relating to the GC peak area of the internal
standard.
In addition, the enzymatic stability of PhLOX was evaluated by using stored
PhLOX (at 4 °C for two weeks) in the incubation of the five pure fatty acids under
standard reaction conditions and observing the declined yield of the volatile products.
RESULTS
Expression, Purification, and Enzymatic Characterization of PhLOX. After
SDSꢀPAGE, the purified PhLOX appeared on the gel as a single band near 100 kDa
(Figure 1), which was consistent with the predicted molecular mass of the deduced
amino acid sequence of PhLOX with an Nꢀterminal His tag (about 98 KDa). The
PhLOX harvested from the total 5 L culture medium amounted to 121.8 mg (i.e., 24.4
mg·L−1), and the final purity reached about 92% as was calculated from the optical
density ratio of the gel.
The enzymatic activity of PhLOX was surveyed using the five PUFAs as
substrates (i.e., LA, αꢀALA, ARA, EPA, and DHA). It was the highest (955.5 U) in
the reaction of EPA (Figure 2A). The EPA substrate was consumed by about 24.7 ꢁM
in mere 6 s and was exhausted in 1 min (Figure 2B). In the case of DHA, the
enzymatic activity of PhLOX reached 807.4 U, and within 5 min 91% of the DHA
substrate had been consumed. In the reaction of ARA, the enzymatic activity of
PhLOX reached 674.4 U and it took 10–15 min to consume 90% of the ARA
substrate. However, the activity of PhLOX was much lower in the reaction of the two
C18 PUFAs (i.e., LA and αꢀALA), and it took as long as 30 min to consume ≥80% of
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the substrate. The results showed that the activity of PhLOX was dramatically higher
in the reaction of the C20 and C22 PUFAs than in the reaction of C18 PUFAs (Figure
2B).
Data were fitted to the Michaelis–Menten model to calculate the kinetic
parameters of the enzymatic reaction of different substrates. It was found that the Vm
of C20 PUFAs was at least 24 times higher than that of C18 PUFAs, and the C22
PUFA (i.e., DHA) also gave a high Vm of 1962.1 ꢁM·min−1 (Table 1). In addition, EPA
was found to be the most suitable substrate among the five tested PUFAs since it gave
a lowest KM of 286.4 ꢁM. The catalytic efficiency (kcat/KM) of PhLOX was also
significantly higher for C20 and C22 PUFAs than for C18 PUFAs. Again, the catalytic
efficiency was the highest when EPA was used as the substrate (kcat/KM = 14.6
min−1·ꢁM−1) (Table 1).
For all substrates, the optimal condition for the enzymatic reaction with PhLOX
was at pH = 8.0 and 20 °C (Figure 3). For the C20 and C22 PUFAs, the activity of
PhLOX remained relatively stable over pH = 6.0–10.0 but decreased sharply when the
pH value was increased to 11.0 or reduced to 2.5. In contrast, for the C18 PUFAs, the
activity of PhLOX was notably affected by the pH value, since only about 20% (for
LA) and 9% (for αꢀALA) enzyme activity was preserved when the pH value increased
from 8.0 to 10.0 (Figure 3B). Similarly, for the C20 and C22 PUFAs, the activity of
PhLOX changed relatively little over the temperature range of 10–40 °C. However,
for the C18 PUFAs, the activity of PhLOX declined sharply when the temperature
deviated from 20 °C (Figure 3A).
Composition of Volatile Products from PUFAs. The volatile products obtained
from the five PUFAs by incubation with PhLOX were analyzed. Data showed that
after incubation with PhLOX, the conversion rate (RS) of five PUFAs were all ≥93%
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and specific volatile organic compounds (VOCs) could be produced from αꢀALA,
ARA, EPA, and DHA, but not from LA (Figures 4 and S4). Note that the products
from ARA included both C8 (1ꢀoctenꢀ3ꢀol, 1ꢀoctenꢀ3ꢀone, 2Zꢀoctenꢀ1ꢀol) and C9
(2Eꢀnonenal) VOCs, whereas EPA and DHA only gave a C9 VOC (2E,6Zꢀnonadienal)
and a C8 VOC (3E,5Zꢀoctadienꢀ2ꢀone), respectively. Among the three C20/C22
PUFAs, ARA gave the highest total VOC yield (74.7 ꢁM, about 75% yield), which
was about 2.5 times and 6.3 times that of EPA and DHA, respectively (Table 2).
Among the C8/C9 VOCs generated from ARA, 2Eꢀnonenal was the primary product,
which amounted to 54.2 ꢁM (about 54% yield). Meanwhile, 2E,6Zꢀnonadienal, also a
C9 VOC, was generated from EPA and amounted to 30.3 ꢁM (about 30% yield). In
addition, various C8 VOCs were generated from ARA and DHA by incubation with
PhLOX. For example, 1ꢀoctenꢀ3ꢀone (9.7 ꢁM) was produced from ARA and
3E,5Zꢀoctadienꢀ2ꢀone (11.8 ꢁM) was produced from DHA. These C8 VOCs were all
obtained in approximately 12% yield. Incubation of αꢀALA with PhLOX generated
minor amounts of C5/C6 VOCs in <5% yield.
Storage of the PhLOX solution at 4 °C for two weeks did not seriously damage
the enzymatic activity. Although 2Zꢀpentenal and 2Zꢀoctenꢀ1ꢀol were detected in trace
amounts in the reactions with stored PhLOX and showed 0 ꢁM after data conversion,
the yield of total VOCs did not decline excessively (Table 2).
Production of VOCs from Hydrolyzed Oils by Incubation with PhLOX.
Three hydrolyzed oils were surveyed as the substrate for the production of VOCs.
Data in the column of ‘before incubation’ showed that hydrolyzed sunflower seed oil
and hydrolyzed corn oil were similar in fatty acid composition, i.e., mainly containing
C18:2 (902.3 ꢁM and 769.1 ꢁM respectively) followed by C18:1 (>24%), together
with minor amount of C18:3 (Table 3). In contrast, hydrolyzed fish oil contained
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many more fatty acids, including C16–C22 PUFAs whose degree of unsaturation
varied from 2 to 6 (Table 4). The most abundant PUFA was C22:6 (471.9 ꢁM),
followed by C20:5 (423.7 ꢁM). There were also minor amounts of C20:4 and C22:5.
The contents of other C16–C18 fatty acids were all less than 10 ꢁM. Although it
seemed that there was a small decline of PUFAs in these oil samples after incubation
with inactive PhLOX as the control, the main fatty acid component was still C18:2 in
two hydrolyzed plant oils (>68%), and the main fatty acids were still C22:6 and C20:5
(45.6% and 41.3%, respectively) in hydrolyzed fish oil (Tables 3 and 4). However,
when the three hydrolyzed oils were used as substrates for incubation with PhLOX, it
was found that only PUFAs were consumed while saturated and monounsaturated
fatty acids remained basically unchanged. This was consistent with the predicted
enzymatic selection of PhLOX. When hydrolyzed sunflower seed oil was used as the
substrate, the minor component C18:3 were completely consumed but the
consumption rate of the major component C18:2 only reached about 66% (586.1 ꢁM).
Similarly, when hydrolyzed corn oil was used as the substrate, only 49.5% C18:2
were consumed, and a minor amount of C18:3 remained. Among the seven PUFAs in
hydrolyzed fish oil, the consumption rate of the major components C20:5 and C22:6
reached 78.8% (322.8 ꢁM) and 87.7% (397.1 ꢁM), respectively, after incubation with
PhLOX. The consumption rate of C18:4, C20:4, and C22:5 was all greater than 75%,
but the absolute amount of these consumed substrates was not high since they were
minor components in the original PUFA profile of hydrolyzed fish oil. It could be seen
that among the PUFAs of different degree of unsaturation that coexisted in hydrolyzed
fish oil, PhLOX preferentially catalyzed PUFAs of high unsaturation (≥4) and barely
targeted the diꢀunsaturated (C18:2 and C16:2) fatty acids. The consumption rate of
C18:2 and C16:2 were very low even though their content in the original PUFA
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mixture was already tiny.
GCꢀMS was used to analyze the new VOCs produced from the three hydrolyzed
oils after incubation with PhLOX. For hydrolyzed sunflower seed oil, six new VOCs
were tentatively identified after incubation with PhLOX, namely 2E,4Eꢀpentadienal,
nꢀheptanal, 2Eꢀheptenal, nꢀoctanal, 2Eꢀoctenal, and 2E,4Eꢀnonadienal (Figures S1 and
S5). The newly generated VOCs were semiꢀquantified to 517.0 ꢁM. Among the new
VOCs, 2E,4Eꢀpentadienal accounted for 70.5% of the total (364.5 ꢁM), followed by
2Eꢀoctenal (102.2 ꢁM) (Table 5). Besides, compared with the control group, the
content of two existing VOCs increased, most notably nꢀnonanal (by 32.1 ꢁM). In the
case of hydrolyzed corn oil, the newly generated VOCs included 2E,4Eꢀpentadienal,
nꢀheptanal, 2Eꢀheptenal, nꢀoctanal, 2Eꢀoctenal, and 2E,4Eꢀnonadienal (Figures S2 and
S5). The major new VOCs were 2E,4Eꢀpentadienal and 2Eꢀoctenal, which amounted
to 69.0% and 23.7% of the total new VOCs, respectively (Table 5). In comparison
with the control group, the amount of nꢀnonanal in the products increased most
notably (from 16.3 ꢁM to 49.0 ꢁM). In addition to the new and increased production
of VOCs above in the two hydrolyzed plant oils, there were still some other VOC
compounds tentatively identified such as 3ꢀpentanone, 2ꢀhexanone, hexanoic acid
methyl ester, glycerin, undecane, octanoic acid methyl ester, dodecane, decanoic acid
methyl ester and dodecanoic acid methyl ester. When hydrolyzed fish oil was used as
the substrate, many more volatile products were tentatively identified, including 13
kinds of C5–C10 VOCs (Figures S3 and S5). Among the newly generated VOCs,
2E,4Eꢀoctadienal had a highest yield of 143.6 ꢁM, which accounted for about 48.5%
of the total new VOCs (Table 5). Meanwhile, 2E,4Zꢀdecadienal and 2E,6Zꢀnonadienal
each also had a high yield of 41.0 ꢁM and 29.3 ꢁM, respectively. In comparison with
the control group, the amount of nꢀnonanal in the products increased most notably
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(i.e., by 48.6 ꢁM, a 4.5ꢀfold increase). Additionally, some other VOC compounds
were tentatively detected, including 3ꢀpentanone, 3ꢀhexanone, 2ꢀhexanone, hexanoic
acid methyl ester, glycerin, 1Hꢀimidazole, octanoic acid methyl ester,
4,6ꢀdimethylꢀdodecane, 2,4ꢀdimethylꢀbenzaldehyde, decanoic acid methyl ester and
dodecanoic acid methyl ester.
DISCUSSION
Since the 1980s, scientists have made considerable efforts to produce volatile
aroma compounds by enzymatic catalysis. Previous studies mainly exploited the
biosynthesis of green leaf volatiles (GLVs) and focused on the associated process
optimization.21,22 Usually, soybean flour is used as the LOX source, and various plant
extracts (e.g., green bell pepper, mint leaves, green leaves from sugar beet) are used as
the HPL source.22 However, the HPL activity of plant extracts appears to be unstable
and tends to be restricted by the catalytic environment. For example, the HPL activity
of plant extracts can be easily inhibited when the concentration of hydroperoxides
becomes unfavorable. In addition, the HPL activity is highly dependent on the source
plant, and the crude extracts may contain other enzymes that can generate unwanted
byproducts.17 Therefore, the development of effective biocatalysts remains a key task
in the biosynthesis of volatile aroma compounds for the industry, e.g., a protein
engineering strategy based on directed evolution to enhance the HPL activity.23 Here,
a new and natural biocatalyst, PhLOX, was found to be suitable for the production of
some VOCs without harsh catalytic conditions because of the inhibition of HPL
activity.
We previously harvested PhLOX from the red alga P. haitanensis and reported on
its multiple functions on fatty acids, i.e., lipoxygenase (LOX), hydroperoxide lyase
(HPL), and allene oxide synthase (AOS).18 This property of PhLOX is not unique;
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some enzymes involved in oxylipin formation have been reported to possess similar,
if not overlapping functions. For example, a catalaseꢀlikeꢀAOS–LOX fusion protein
from coral Plexaura homomalla converts arachidonic acid completely to an allene
oxide.24 PpLOX1 from moss Physcomitrella patens shows additional HPL activity to
produce 2Zꢀoctenꢀ1ꢀol and 1ꢀoctenꢀ3ꢀol from arachidonic acid.25 However, compared
with these fused lipoxygenases, the quite exceptional trait of PhLOX was its
LOXꢀAOSꢀHPL activities in one catalytic domain which belongs to a conserved
nonꢀheme LOX family. And unlike some HPL enzymes harvested from some
plants,26,27 PhLOX does not contain a Nꢀterminal transit peptide. Hence, there was no
insertion of the recombinant enzyme in the membrane during expression in E. coli,
and a larger amount of the enzyme could thus be recovered in the soluble protein
fraction. Under the current conditions, the yield of PhLOX from cell lysis reached
24.4 mg protein per 1 L culture, which is much higher than the yield of some
recombinant plant HPL, such as recombinant olive HPLwt (about 1.3 mg·L−1).15
Because of its HPL activity, the PhLOX was used as a novel biocatalyst in this
study for the oneꢀstep conversion of different PUFAs or hydrolyzed oils into VOCs.
Among the five tested PUFAs (i.e., LA, αꢀALA, ARA, EPA, and DHA), it was found
that under identical reaction conditions, the activity of PhLOX was higher when C20
and C22 PUFAs, rather than C18 PUFAs, were used as the substrate. The
consumption rate of C20 and C22 PUFAs was also higher than that of C18 PUFAs.
According to the Michaelis constant (KM), the most suitable substrate for PhLOX was
EPA. Besides, PhLOX clearly preferred fatty acids with high degree of unsaturation
(≥3) as the substrate, since the KM of LA (459.1 ꢁM) was much higher than that of
EPA and DHA (286.4 and 316.3 ꢁM, respectively). The catalytic efficiency of PhLOX
was also higher when C20 and C22 PUFAs were used as the substrate, as was
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indicated by the higher kcat/KM value of ARA, EPA and DHA than that of LA and
αꢀALA. These results are in agreement with previous findings. It was reported that the
LOXs of Rhodophyta used both octadecenoic acids and eicosenoic acids as
substrates.28 Andreou et al. pointed out that the most prominent theme in red algal
oxylipin biosynthesis was the metabolism of eicosenoic acids.29 In our case, we also
noted that when C20 and C22 PUFAs were used as substrates, the activity of PhLOX
remained stable over a broad range of pH (6.0–10.0) and temperature (10–40 °C).
Therefore, it would be desirable for industrial scale synthesis to use biological oils
that are rich in C20 and C22 PUFAs when PhLOX is used as the catalyst.
Analysis of the VOC products from the PUFAs also showed that PhLOX
preferably catalyzed the conversion of PUFAs with high degree of unsaturation (>3)
into C8 and C9 VOCs. In the case of C18 PUFA substrates, PhLOX did not convert
LA into VOCs and converted αꢀALA only into a slight amount of C5 (2Zꢀpentenal)
and C6 (3Zꢀhexenal) VOCs. It can be deduced that 2Zꢀpentenal and 3Zꢀhexenal were
generated from the cleavage of 13ꢀhydroperoxyoctadecatrienoic acid (13ꢀHpOTE) at
ωꢀC5 and ωꢀC6, respectively. Although oxidation also happened at ωꢀC10 and
9ꢀHpOTE was detected, PhLOX did not help to generate C9 aldehydes from αꢀALA.18
This differed from some vegetable HPL that are known to convert 9ꢀHpOTE or
9ꢀhydroperoxyoctadecadienoic acid (9ꢀHpODE) into C9 aldehydes (e.g.,
2E,6Zꢀnonadienal and 2Eꢀnonenal).30 Furthermore, C5/C6 VOCs were obtained from
αꢀALA in a very poor yield of 4.5%. In contrast, the yield of C6/C9 VOCs was within
25%–86% when plant extracts were used as the HPL source,22 or about 93% for
hexanal production and 73% for 3Zꢀhexenal production when olive recombinant HPL
was used as the catalyst.15
However, when C20 and C22 PUFAs were used as substrates, PhLOX evidently
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catalyzed the formation of VOCs. Incubation of ARA with PhLOX produced both C8
and C9 VOCs, including 1ꢀoctenꢀ3ꢀol, 1ꢀoctenꢀ3ꢀone, 2Zꢀoctenꢀ1ꢀol, and 2Eꢀnonenal,
and the total yield reached as high as 74.7%. Among these compounds, the three C8
VOCs, which contributed significantly to the aroma of mushroom,31 were formed by
the cleavage at ωꢀC8 of 12ꢀhydroperoxyeicosatetraenoic acid (12ꢀHpETE). Previous
reported biosynthesis of 1ꢀoctenꢀ3ꢀol/one mainly proceeded via the aerobic oxidation
of linoleic acid using LOX and HPL.11,32 Hence, the synthesis of 1ꢀoctenꢀ3ꢀol/one
from ARA with PhLOX as the biocatalyst could serve as an alternative strategy. In
fact, in the PhLOX catalyzed reaction, the predominant volatile product from ARA
was the C9 aldehyde 2Eꢀnonenal (in 54.2% yield). Forss et al. reported that
2Eꢀnonenal was a major flavor compound of cucumber and could be converted from
linoleic acid via 9ꢀHpODE during biosynthesis.33 However, in the PhLOX catalyzed
reaction, 2Eꢀnonenal was generated by the cleavage of 12ꢀHpETE at ωꢀC9. The
current PhLOX reaction may provide a feasible pathway for the industrial production
of 2Eꢀnonenal in high yield.
Incubation of EPA and DHA with PhLOX generated 2E,6Zꢀnonadienal and
3E,5Zꢀoctadienꢀ2ꢀone as the only volatile product, respectively. Kocsy et al. reported
that 2E,6Zꢀnonadienal gave cucumber flavor.30 Hu and Pan also reported that
2E,6Zꢀnonadienal had a fresh fishꢀlike scent, and 3E,5Zꢀoctadienꢀ2ꢀone had a
melonꢀlike flavor.34 Currently, there are relatively few studies on enzymatic catalysis
for the synthesis of 2E,6Zꢀnonadienal and 3E,5Zꢀoctadienꢀ2ꢀone as food aroma
compounds. In this work, incubation of EPA and DHA with PhLOX generated
12ꢀhydroperoxyeicosapentaenoic
acid
(12ꢀHpEPE)
and
14ꢀhydroperoxy ocosahexaenoic acid (14ꢀHpDHE), and cleavage at ωꢀC9 of
d
12ꢀHpEPE and at ωꢀC8 of 14ꢀHpDHE then gave rise to 2E,6Zꢀnonadienal and
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3E,5Zꢀoctadienꢀ2ꢀone, respectively. The results here are noteworthy since PhLOX is
the first enzyme reported in literature that can use C20/C22 PUFAs as substrates to
generate C8/C9 VOCs in one step and on its own.
Hydrolyzed oils were also tested as substrates and incubated with PhLOX to
produce VOCs. During enzymatic incubation, most PUFAs components were
transformed by PhLOX, and only a minor fraction was degraded by spontaneous
oxidation. In the case of hydrolyzed sunflower seed oil and hydrolyzed corn oil,
although ≥50% of C18:2 were metabolized by PhLOX, no corresponding VOCs were
produced. Because the degradation of αꢀALA by PhLOX produced a low yield (about
4.5%) of C5/C6 VOCs (i.e., 2Zꢀpentenal and 3Zꢀhexenal), it is reasonable that no
2Zꢀpentenal nor 3Zꢀhexenal were detected when hydrolyzed sunflower seed oil and
corn oil were treated with PhLOX since only about 0.4 ꢁM and 9.9 ꢁM C18:3 were
consumed. Instead, an isomer of 3Zꢀhexenal, 2Eꢀhexenal, was produced. Németh et al.
reported that both 2Eꢀhexenal and 3Zꢀhexenal were formed from hydrolyzed linseed
oil by using soybean LOX1 and green bell pepper homogenate.21 However, it was
unexpected that two volatile compounds, 2E,4Eꢀpentadienal and 2Eꢀoctenal, were
generated in high concentrations and four other VOCs were formed in low
concentrations, by comparing with the control group of incubation with inactive
PhLOX. It has been reported that some VOC compounds including 1ꢀpentenꢀ3ꢀone,
hexanal, 1ꢀpentanol, octanal, 1ꢀoctenꢀ3ꢀone, 1ꢀoctenꢀ3ꢀol, and 2E,4Zꢀheptadienal were
crucial to vegetable oil quality.35,36 For example, Wei et al. reported that some
characteristic aroma compounds such as 2E,4Eꢀpentadienal (green, oily),
2E,4Eꢀheptadienal (sweet, hazelnut, woody) and 5ꢀethyldihydroꢀ2(3H)ꢀfuranone
(cerealꢀlike) were contained in Xinjiang flaxseed oil (China), although information on
flaxseed oil volatiles remains limited.19 In our experiments, both sunflower seed oil
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and corn oil were hydrolyzed and extracted for use as substrates of PhLOX. GCꢀMS
analysis showed that there were still esters and alkanes at high levels in the control
group incubated with inactive PhLOX. However, after incubation with PhLOX, more
VOCs appeared in both plant oil samples, suggesting that PhLOX could improve and
reconstruct the aroma composition of plant oils. In the food industry, both
2E,4Eꢀpentadienal and 2Eꢀoctenal are permitted for use as flavor additives in candy,
beverage, meat, etc., since 2E,4Eꢀpentadienal has the flavor of fruit and 2Eꢀoctenal
provides the scent of fat. Currently, these two compounds are mostly obtained through
chemical synthesis in industry. The high yield of 2E,4Eꢀpentadienal and 2Eꢀoctenal
from vegetable oils by incubation with PhLOX indicates that this reaction may be a
potentially viable biosynthesis in food industry for these aroma compounds.
When hydrolyzed fish oil was incubated with PhLOX, the enzyme preferentially
targeted PUFAs with high degree of unsaturation, since the consumed substrates were
mainly C18–C22 PUFAs with a degree of unsaturation no less than 4. Nevertheless,
although the consumed substrates were mainly C20:5 and C22:6, the primary new
VOC was neither 2E,6Zꢀnonadienal nor 3E,5Zꢀoctadienꢀ2ꢀone, but 2E,4Eꢀoctadienal.
When C20/C22ꢀPUFAs were incubated with PhLOX, the yield of 2E,6Zꢀnonadienal
from EPA and 3E,5Zꢀoctadienꢀ2ꢀone from DHA were about 30% and 12%,
respectively. When hydrolyzed fish oil was incubated with PhLOX, the formed
2E,6Zꢀnonadienal and 3E,5Zꢀoctadienꢀ2ꢀone were about 29 ꢁM and 17 ꢁM,
respectively, from 322 ꢁM and 397 ꢁM of C20:5 and C22:6 , with yields of 9.0% and
4.3%. These yields were one third of that of EPA and DHA. In addition, although the
yield of 2Eꢀnonenal and C8ꢀVOCs (i.e., 1ꢀoctenꢀ3ꢀol, 1ꢀoctenꢀ3ꢀone and
2Zꢀoctenꢀ1ꢀol) from ARA by PhLOX was about 54% and 20.5%, respectively, no
2Eꢀnonenal but a minor amount of 2Eꢀoctenꢀ1ꢀol was detected when fish oil
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hydrolysate containing 36 ꢁM C20:4 was treated with PhLOX. It seems that some
components in hydrolyzed fish oil interfered with the activity of PhLOX, which was
also observed when hydrolyzed vegetable oils were used as substrates for PhLOX.
Reports have proved that except the rich unsaturated fatty acid components in plant
oils, there were a portion of phytosterols, alcohols, vitamins, polyphenols.37 It may be
appealing to study further the catalyzed metabolism of PhLOX, considering its quite
exceptional trait as holding LOXꢀAOSꢀHPL activities in one nonꢀheme catalytic
domain.18 Hu and Pan reported that incubating fish oil with the crude extract of a
green marine macroalga Ulva conglobate could increase desirable unsaturated
aldehydes, ketones, and alcohols, such as 2E,4Eꢀoctadienal (oyster flavor),
2E,6Zꢀnonadienal (fresh fish flavor), 2Eꢀhexenal (apple flavor), 3E,5Eꢀoctadienꢀ2ꢀone
(fruit flavor), etc.34 In this work, in addition to giving the major product
2E,4Eꢀoctadienal, the incubation reaction with PhLOX also yielded
2E,4Eꢀpentadienal,
2Eꢀhexenal,
4Zꢀheptenal,
1ꢀheptenꢀ3ꢀol,
2Eꢀoctenal,
2E,4Zꢀdecadienal, etc. Hence, using PhLOX as the catalyst for incubation with fish oil
may produce fresh seafood flavors and improve the flavor of the oil itself.
In summary, we used a multifunctional enzyme PhLOX to realize the oneꢀstep
biosynthesis of C8–C9 volatile aroma compounds. The enzyme PhLOX was found to
have high efficiency, high stability, and broad tolerance to reaction conditions. It
could work with a variety of substrates and thus could serve as a good catalyst to
produce C8ꢀ and C9ꢀVOCs, especially 1ꢀoctenꢀ3ꢀol/one, 2Eꢀnonenal,
2E,6Zꢀnonadienal, and 3E,5Zꢀoctadienꢀ2ꢀone. Hydrolyzed oils (sunflower seed oil,
corn oil, and fish oil) could also be incubated with PhLOX to produce
2E,4Eꢀpentadienal, 2Eꢀoctenal, and 2E,4Eꢀoctadienal. It is possible that PhLOX may
be further combined with lipase in the oneꢀstep bioconversion of fatty acids to
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produce aroma compounds of more appealing flavor.
ASSOCIATED CONTENT
Supporting Information
GCꢀMS analysis of the VOCs composition of hydrolyzed oils by incubation with
PhLOX.
AUTHOR INFORMATION
Corresponding authors
*Tel.:
+86ꢀ0574ꢀ87609572.
Fax:
Fax:
+86ꢀ0574ꢀ87609570.
+86ꢀ0574ꢀ87609581.
EꢀMail:
EꢀMail:
chenhaimin@nbu.edu.cn.
*Tel.:
+86ꢀ0574ꢀ87600738.
xiaojunyan@hotmail.com.
Funding
This project was funded by the NSFC project (No. 81370532), the Major Scientific
and Technological Project of Zhejiang Province (2016C02055ꢀ6B), National Science
Foundation of Zhejiang (LY18C190004), Ningbo Programs for Science and
Technology Development (2017C110026, 2017C10020), China Agriculture Research
System (CARꢀ50), K.C. Wong Magna Fund of Ningbo University, the 151 talents
Project, the Li Dak Sum Marine Biopharmaceutical Development Fund, and the
National 111 Project of China.
Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Schultes, R. E. Common Fragrance and Flavor Materials: Preparation, Properties
and Uses. Economic Botany 1987, 41 (4), 493ꢀ493.
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Figure legends
Figure 1. SDSꢀPAGE analysis of purified PhLOX.
Figure 2. Analysis of enzymatic activity when different fatty acids were used as
substrate for incubation with PhLOX. (A) Enzymatic activity of PhLOX at 1 min for
the reaction of five different fatty acid substrates. (B) Consumption of substrate over
time.
Figure 3. Analysis of the impact of (A) temperature and (B) pH on the incubation
reaction with PhLOX. The tested substrates included linoleic acid (LA), αꢀlinolenic
acid (αꢀALA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), and
docosahexaenoic acid (DHA). The curves were plotted based on GaussAmp fitness.
Figure 4. GCꢀMS analysis of VOCs generated from different fatty acids after
incubation with PhLOX.
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