Characterization of 9-fatty acid hydroperoxide lyase-like activity in germinating barley seeds that transforms 9(S)-hydroperoxy-10(E),12(Z)- octadecadienoic acid into 2(E)-nonenal

Article abstract of DOI:10.1271/bbb.69.1661

Previously, we reported that 2(E)-nonenal, having a low flavor threshold (0.1 ppb) and known as the major contributor to a cardboard flavor (stale flavor) in stored beer, is produced by lipoxygenase-1 and a newly found factor named 9-fatty acid hydroperoxide lyase-like (9-HPL-like) activity in malt. To assess the involvement of 9-HPL-like activity in beer staling, we compared the values of the wort nonenal potential, an index for predicting the staleness of beer, with the lipoxygenase and 9-HPL-like activity of 20 commercial malts. There was a significant correlation between the malt 9-HPL-like activity and the values of wort nonenal potential (r = 0.53, P < 0.05), while the correlation between malt lipoxygenase activity and the wort nonenal potential was statistically insignificant. Analysis of the partially purified 9-HPL-like activity from embryos of germinating barley seeds indicated that 9-HPL-like activity consisted of fatty acid hydroperoxide lyase and 3Z:2E isomerase.

Full text of DOI:10.1271/bbb.69.1661

Biosci. Biotechnol. Biochem., 69 (9), 1661–1668, 2005
Characterization of 9-Fatty Acid Hydroperoxide Lyase-Like
Activity in Germinating Barley Seeds That Transforms
9(S)-Hydroperoxy-10(E),12(Z)-octadecadienoic Acid into 2(E)-Nonenal
Hisao KURODA,y Hidetoshi KOJIMA, Hirotaka KANEDA, and Masachika TAKASHIO
Frontier Laboratories of Value Creation, SAPPORO BREWERIES LTD.,
10 Okatohme, Yaizu, Shizuoka 425-0013, Japan
Received December 15, 2004; Accepted July 1, 2005
Previously, we reported that 2(E)-nonenal, having a
low flavor threshold (0.1 ppb) and known as the major
contributor to a cardboard flavor (stale flavor) in stored
beer, is produced by lipoxygenase-1 and a newly found
factor named 9-fatty acid hydroperoxide lyase-like (9-
HPL-like) activity in malt. To assess the involvement of
9-HPL-like activity in beer staling, we compared the
values of the wort nonenal potential, an index for
predicting the staleness of beer, with the lipoxygenase
and 9-HPL-like activity of 20 commercial malts. There
was a significant correlation between the malt 9-HPL-
like activity and the values of wort nonenal potential
(r ¼ 0:53, P < 0:05), while the correlation between malt
lipoxygenase activity and the wort nonenal potential was
statistically insignificant. Analysis of the partially puri-
fied 9-HPL-like activity from embryos of germinating
barley seeds indicated that 9-HPL-like activity consisted
of fatty acid hydroperoxide lyase and 3Z:2E isomerase.
forcing test to measure the potential of wort to form
2(E)-nonenal. The values are expressed as the concen-
tration (ppb) of 2(E)-nonenal in the heat-treated wort
samples. Very successful malt selection and malting
methods for improving the flavor stability of beer by
reducing the values of the wort nonenal potential have
been developed.4,5) Although we often use 2(E)-nonenal
as a marker for beer staling, the biochemical background
of the formation of 2(E)-nonenal during mashing is not
fully understood. Therefore, in a previous study, we
characterized the factors involved in the production of
2(E)-nonenal during mashing and found that 2(E)-
nonenal is produced by the cascade reaction of barley
lipoxygenase-1 (LOX-1) and malt 9-fatty acid hydro-
peroxide lyase-like activity (9-HPL-like activity) in
malt.6) 9(S)-Hydroperoxy-10(E),12(Z)-octadecadienoic
acid (9-HPOD) produced by LOX-1 was further cleaved
by 9-HPL-like activity to generate 2(E)-nonenal during
mashing. 9-HPL-like activity was like that of an enzyme
because it was sensitive to proteinase-K treatment, and
was moreover completely terminated when boiled. Malt
also contained heat-labile 13-fatty acid hydroperoxide
lyase-like activity (13-HPL-like activity) that trans-
formed 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic
acid (13-HPOD) into hexanal. The 9- and 13-HPL-like
activities were considered to be distinct factors, because
they had different sensitivity to proteinase-K.
Fatty acid hydroperoxide lyase (HPL), ubiquitously
found in the plant kingdom, catalyzes the cleavage
reaction of fatty acid hydroperoxides into oxo-acids and
aldehydes. Intensive studies on HPL in dicots have
revealed that HPL is a member of the cytochrome P450
family, which plays an important role in the interaction
among plant-herbivores by producing phytooxylipins.7,8)
HPL is grouped by substrate specificity into two groups,
13-HPL (CYP74B) and 9-/13-HPL (CYP74C).9,10) 9-/
13-HPL cleaves 9-HPOD, 9(S)-hydroperoxy-10(E),
12(Z), 15(Z)-octadecatrienoic acid, 13-HPOD, and
13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid
Key words: fatty acid hydroperoxide lyase; barley; 2(E)-
nonenal; 9(S)-hydroperoxy-10(E),12(Z)-
octadecadienoic acid; 3Z:2E isomerase
Flavor-active compounds derived from oxygenated
lipids have a large impact on the quality of foods and
beverages.1) Malt contains lipids and lipid-oxidizing
enzymes, which in turn produce flavor-active oxygen-
ated species during mashing. Among them, 2(E)-
nonenal has a very low flavor threshold in beer
(0.7 nM, 0.1 ppb),2) and is considered to be the major
contributor to the ‘‘cardboard flavor’’ that arises when a
beer is stored at high temperature or for a prolonged
period. There is correlation among malt lipoxygenase
(LOX), the wort nonenal potential, 2(E)-nonenal that
develops in beer after storage, and the sensory scores of
staleness characterizing the beer as having ‘‘cardboard
flavor’’.3) The test of wort nonenal potential, widely used
by brewers and brewing chemists to estimate the quality
of malt related to beer flavor stability, is a kind of
y
To whom correspondence should be addressed. Fax: +81-54-629-3144; E-mail: Hisao.Kuroda@sapporobeer.co.jp
Abbreviations: HPL, fatty acid hydroperoxide lyase; 9-HPOD, 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid; 13-HPOD, 13(S)-hydro-
peroxy-9(Z),11(E)-octadecadienoic acid; LOX, lipoxygenase

1662
H. KURODA et al.
into 3(Z)-nonenal, 3(Z),6(Z)-nonadienal, hexanal, and
3(Z)-hexenal respectively. On the other hand, 13-HPL
preferentially cleaves 13-HPOD and 13(S)-hydroperoxy-
9(Z),11(E),15(Z)-octadecatrienoic acid into hexanal and
3(Z)-hexenal respectively. 3(Z)-Nonenal, 3(Z),6(Z)-
nonandienal, and 3(Z)-hexenal are spontaneously or
enzymatically isomerized into 2(E)-nonenal, 2(E),6(Z)-
nonadienal, and 2(E)-hexenal respectively. HPL affects
the flavor characteristics of food products by changing
the constituents of volatile aldehydes.11–13) Although
there are several studies on the effect of dicot HPL on
food quality, in monocots, especially in cereal products
including malt, there have been few studies on the
presence of HPL activity, and none on its role in the
production of volatile aldehydes in foods until our
report.6) The only published report to date is on the
presence of the cDNA clone that encodes for 13-HPL in
barley. The recombinant protein of this cDNA expressed
in E. coli was shown to cleave only 13-HPOD and 13-
HPOT into hexanal and hexenal, but had no activity
against 9-HPOD or 9-HPOT,14) so that information on
the fatty acid hydroperoxide lyase that catalyzes the
transformation of 9-HPOD and 9-HPOT in monocots is
still missing. The 9-HPL-like activity found in malt is
considered to be possibly the first HPL that cleaves
9-HPOD in monocots.
In this study, we first analyzed the correlation
between 9-HPL-like activity in malt and the values of
the wort nonenal potential. Secondly, we partially
purified and characterized the biochemical properties
of 9-HPL-like activity from embryos of germinating
barley seeds. There was significant correlation between
9-HPL-like activity and the wort nonenal potential,
which implies the involvement of 9-HPL-like activity in
the formation of cardboard flavor. Biochemical analysis
revealed that 9-HPL-like activity consisted of HPL and
3Z:2E isomerase. Our study indicates the presence of
HPL that cleaves 9-HPOD in monocots and its impor-
tance for the qualities of foods and beverages made from
cereals.
The sample was dissolved in 10 ml of hexane/ether (9:1)
and applied to a silica-gel column (silica-gel 60, Merk;
particle size, 0.063–0.2 mm; column size, 1 cm in
diameter ꢁ 5 cm long) that was pre-equilibrated with
hexane/ether (9:1). After the column was washed with
60 ml of hexane/ether (9:1), 9-HPOD was eluted with
hexane/ether (1:1) and fractionated. The fractions were
analyzed with TLC and then visualized with both UV
and iodide. Fractions containing 9-HPOD free from
linoleic acid were collected, and the solvent was dried
under nitrogen. 9-HPOD was dissolved in ethanol at a
concentration of 20 mM and stored in aliquots at
ꢂ135 ꢀC until use. The purity of 9-HPOD was deter-
mined using straight-phase HPLC as described previ-
ously.6)
Enzyme assays of 9-HPL-like activity and 3Z:2E
isomerase. 9-HPL-like activity was measured as fol-
lows. The enzyme solution was diluted with 0.1 M Tris–
HCl (pH 7.0) to 100 ml in an eppendorf tube, and 5 ml of
2 mM 9-HPOD dissolved in ethanol was added. The
reaction mixture was incubated at 30 ꢀC for 5 min and
the reaction was stopped by the addition of ice-cold
ethanol containing 40 ppb 1-nonanol (0.9 ml). After
chilling on ice for 5 min, it was centrifuged at 10;000 ꢁ
g at 4 ꢀC for 1 min. The supernatant was transferred to a
vial containing 6.5 ml of distilled water and 3 g of
sodium chloride and sealed with a silicon-coated cap.
2(E)-Nonenal was trapped by inserting a SPME fiber
(100 mm membrane-thick polydimethylsiloxane solid
phase micro extraction fiber probe, Spelco, PA,
U.S.A.) into the headspace of the vial at 40 ꢀC for
15 min. The probe was then introduced into the injection
port and thermally desorbed at 260 ꢀC for 5 min onto a
DB-1 column (liquid phase = dimethylpolysiloxane,
30 m ꢁ 0:25 mm, film thickness = 1 mm, J&W Scientif-
ic, CA, U.S.A.). GC–MS analysis was conducted with
the Hewlett Packard HP6890/MSD system. The select-
ed ions used were m=z ¼ 69 and 70. The flow rate of
helium gas was 1.0 ml/min. The temperature of the oven
was maintained at 60 ꢀC for 1 min, and then raised by
5 ꢀC/min to 260 ꢀC. Calibration curves were derived
from the authentic 2(E)-nonenal solution containing the
internal standard (1-nonanol). The concentration of
2(E)-nonenal in each sample was measured by the peak
area relative to that of the internal standard. 9-HPL-like
activity was expressed as the rate of formation of 2(E)-
nonenal (in katal).
Materials and Methods
Chemicals. Nordihydroguiaretic acid was from ICN
Biomedicals (Ohio, U.S.A.). Salicylic acid, 2(E)-none-
nal, and hexanal were from Wako Pure Chemical
Industries (Osaka, Japan). 3(Z)-Nonenal was from
Larodan Fine Chemicals (Malmo, Sweden). 9(S)-Hydro-
¨
peroxy-10(E),12(Z)-octadecadienoic acid (9-HPOD)
was produced through the reaction of recombinant
barley LOX-1 (rLOX-1)15) and linoleic acid as follows:
rLOX-1 (22 mkat) was diluted with 300 ml of 0.1 M
sodium acetate buffer (pH 6.0) and incubated with
15 ml of 40 mM of linoleic acid dissolved in ethanol at
20 ꢀC for 5 min. The reaction was terminated by adding
30 ml of CH2Cl2 and 25 ml of 5 M NaCl, and the mixture
was vigorously shaken. After standing for 10 min, the
CH2Cl2 layer was recovered and dried under nitrogen.
3Z:2E Isomerase activity was measured as follows.
The enzyme solution was diluted with 0.1 M Tris–HCl
(pH 7.0) to 100 ml in an eppendorf tube and 5 ml of 2 mM
3(Z)-nonenal (ethanol solution) was added. After the
reaction mixture was incubated at 30 ꢀC for 20 min,
products were extracted with 200 ml of ether containing
50 ppb 1-nonanol as an internal standard. A portion of
ether layer (2 ml) was subjected directly onto GC
equipped with the same column as previously described.
GC–MS analysis was conducted similarly, except for the

Characterization of 9-Fatty Acid Hydroperoxide Lyase-Like Activity
1663
temperature program (120 ꢀC for 1 min, then raised by
5 ꢀC/min to 150 ꢀC, and finally 40 ꢀC/min to 300 ꢀC).
The amount of 2(E)-nonenal was measured as described.
3Z:2E isomerase activity was expressed as a rate of the
formation of 2(E)-nonenal (in katal).
inserted either immediately, or after incubation at 30 ꢀC
for 5 min. Aldehydes accumulated in the vial were
trapped with SPME fiber at 30 ꢀC for 5 min and
subjected to GC–MS. The condition of GC oven was
same as that for the 3Z:2E isomerase assay. Products
were identified by mass-spectrum analysis and the
coincidence of retention time with authentic compounds.
To analyze the substrate effect on velocity, 9-HPL-
like activity was assayed at final concentrations of
0.008–0.84 mM with concentrated (ꢁ20) 9-HPOD dis-
solved in ethanol. The apparent Km value was estimated
by Lineweaver–Burk reciprocal plots. To test the effect
of salicylic and nordihydroguiaretic acids on 9-HPL-like
activity, concentrated solutions (ꢁ20) were added to the
reaction mixture at a final concentration of 0.1 mM or
1 mM. To analyze the effect of pH, 0.1 M potassium
phosphate buffer (pH 5.0–8.0), 0.1 M 2-(N-morpholino)-
ethanesulfonic acid (MES)–KOH (pH 5.5–6.5), and
0.1 M Tris–HCl (pH 8.0) were used instead of 0.1 M
Tris–HCl (pH 7.0).
The molecular weight of 9-HPL-like activity was
estimated by gel–chromatography. A portion of partially
purified 9-HPL-like activity (4 pkat) was run on a
Superdex 200 HR 10/30 gel filtration column (Amer-
sham Pharmacia Biotech; bed diameter = 10 mm,
length = 300 mm, bed volume = 24 ml) using an
AKTA purifier FPLC system (Amersham Pharmacia
Biotech) at 20 ꢀC, and fractions of 0.5 ml were collected
and assayed for 9-HPL-like activity. The marker
proteins (all from Amersham Pharmacia Biotech) used
were ovalbumin (hen’s egg, 43 kDa), albumin (bovine
serum, 67 kDa), aldolase (rabbit muscle, 158 kDa), and
catalase (bovine liver, 232 kDa). The elution profiles
were analyzed with Unicorn software, Version 4
(Amersham Pharmacia Biotech).
Analysis of 9-HPL-like activity, lipoxygenase and
nonenal potential of 20 commercial malts. A total of 20
commercial malts from domestic, European, and Aus-
tralian varieties were used for the analysis. Malt enzyme
extract was prepared as previously reported.6) Briefly,
malt meal (1 g) was stirred with 10 ml of 0.1 M
potassium phosphate buffer (pH 6.0), 0.15% TritonX-
100 for 1 h at room temperature. After centrifuging at
10;000 ꢁ g for 20 min, the supernatant was used as an
enzyme extract. 9-HPL-like activity was measured as
described. LOX activity was measured spectrophoto-
metrically as previously reported.6) LOX activity (in
katal) was expressed as the rate of formation of
conjugated diene chromophore (" ¼ 25;000). Wort was
prepared using the method of the European Brewery
Convention,16) using a Lochner LB8 mashing machine
¨
(Lochner, Germany). The wort nonenal potential was
¨
determined using Drost’s method.3) Statistical analysis
was carried out with a SPSS 9.0J for Windows software
(SPSS Japan, Tokyo, Japan).
Partial purification of 9-HPL-like activity. One gram
of embryos was excised from germinating barley seeds
(Hordeum vulgare, cv. Haruna nijo), and homogenated
in 10 ml of ice-cold 0.1 M sodium acetate, 5 mM EDTA,
0.1% Tween20, 5 mM DTT, and EDTA-free Complete
protease inhibitorꢀ (Roche, Basel, Switzerland), pH
5.5. After centrifuging at 10;000 ꢁ g at 4 ꢀC for 1 h, the
supernatant was desalted with a Hitrap desalting column
(Amersham Pharmacia Biotech) into 10 mM Tris–HCl
(pH 7.2), 0.1% Tween 20, and 1 mM ꢀ-mercaptoethanol.
An eluent was applied to a Resource Q column
(Amersham Pharmacia Biotech; diameter ¼ 6:4 mm,
length ¼ 30 mm, bed volume = 1 ml) equilibrated with
10 mM Tris–HCl (pH 7.2), 0.1% Tween20, and 1 mM ꢀ-
mercaptoethanol, and eluted by a NaCl gradient (0–1 M),
using an AKTA purifier FPLC system (Amersham
Pharmacia Biotech) at 4 ꢀC. Fractions of 0.5 ml were
collected and assayed for 9-HPL-like activity. 9-HPL-
like activity was also purified with Mono-P 5/50 GL
chromatofocusing chromatography (Amersham Pharma-
cia Biotech), Resource S cation-exchange chromatog-
raphy (1 ml) (Amersham Pharmacia Biotech), or
Hydroxyapatite Bio-Gel HT Gel chromatography (Bio-
Rad Laboratories, U.S.A.).
Substrate specificity was determined spectrophoto-
metrically by monitoring a decrease in the substrates as
follows: 9-HPOD or 13-HPOD was diluted to 1 ml of
0.1 M Tris–HCl (pH 7.0) in a cell at a final concentration
of 40 mM. An aliquot of the partial purified fraction was
mixed, and absorbance at 234 nm was monitored at
room temperature.
Results
Analysis of correlation between wort nonenal poten-
tial (beer staling index), 9-HPL-like activity, and the
LOX activity of malt
To examine the relation between 9-HPL-like activity
and malt quality with regard to beer staling, the
enzymatic activities of 9-HPL-like activity, malt LOX
activity, and the values of the wort nonenal potential of
20 randomly sampled commercial malts were compared.
We observed the production of 2(E)-nonenal but not
3(Z)-nonenal when malt extracts were incubated with 9-
HPOD. The wort nonenal potential, an index widely
used by brewers and brewing chemists to evaluate the
quality of malt related to beer staling, is a kind of
Characterization of 9-HPL-like activity. GC–MS
analysis of aldehydes produced by 9-HPL-like activity
was as follows: Partially purified 9-HPL-like activity
(30 mg) was diluted to 1 ml of 0.1 M Tris–HCl (pH 7.0)
in a GC vial and mixed with 9-HPOD at a final
concentration of 0.1 mM. After capping, SPME fiber was

1664
H. KURODA et al.
Fig. 1. Correlation between the Wort Nonenal Potential (Beer Staling Index), 9-HPL-Like Activity, and LOX Activity of 20 Commercial Malts.
The wort nonenal potential, 9-HPL-like activity, and LOX activity were measured for 20 commercial malts. The correlation between the malt
9-HPL-like activity and the values of wort nonenal potential was statistically significant (r ¼ 0:53, P < 0:05 by Pearson’s correlation
coefficient). Malt A, with half the wort nonenal potential of Malt B, had three times more LOX activity, while Malt A had 2.5 times less 9-HPL-
like activity. Symbols: A, Malt A; B, Malt B.
forcing test to measure the potential of wort to form
2(E)-nonenal; the values are expressed as the concen-
tration of 2(E)-nonenal in heat-treated wort samples.3)
There is abundant evidence that the values of the wort
nonenal potential correlate positively with the formation
of 2(E)-nonenal after the beer storage, sensory scores of
staleness of beer, and how much a beer is described as
having a ‘‘cardboard flavor’’.4,5) As clearly shown in
Fig. 1, there was a significant correlation between the
malt 9-HPL-like activity and the values of the wort
nonenal potential (r ¼ 0:53, P < 0:05 by Pearson’s
correlation coefficient). In contrast, the correlation
between malt LOX activity and the wort nonenal
potential was statistically insignificant. However, since
there are two isozymes (LOX-1 and LOX-2) in
germinating barley seeds,15) and in the present study
we measured only the total LOX activity, we might
observe significant correlation between malt LOX-1 and
the wort nonenal potential if we specifically measure
malt LOX-1 activity. It is interesting that ‘‘Malt A’’ with
half the wort nonenal potential of ‘‘Malt B’’ had three
times more LOX activity. On the other hand, ‘‘Malt A’’
had 2.5 less 9-HPL-like activity than ‘‘Malt B’’. This
implies that although the levels of 9-HPL-like activity
(0.033–0.103 nkat/g) were much lower than those of
LOX activity (detection limit to 21 nkat/g) in malt,
9-HPL-like activity might be the determining factor of
the values of wort nonenal potential.
ment showed that 9-HPL-like activity was localized only
in embryos, and not elsewhere in the germinating barley
seeds. Differently from the enzyme extract of malt,
dilution of an extract from embryos resulted in an
increase in the specific activity of 9-HPL-like activity;
afterwards, we confirmed that this was due to the
presence of inhibitory factors. This effect was observed
for both the crude extract and the fraction obtained by
gel-filtration (Mr > 5;000). Partial purification by Re-
source Q anion-exchange column chromatography led to
a single active-peak fraction of 9-HPL-like activity that
was free from inhibitory factors (Fig. 2). The dilution
effect was observed no more in this sample. We
confirmed that the production of 2(E)-nonenal increases
linearly with increases in enzyme extract. In no fraction
was production of 3(Z)-nonenal observed.
This partially purified fraction contained both HPL
and 3Z:2E isomerase. Figure 3A shows the GC–MS
analysis (total ion chromatograms) of the products
formed by incubation of 9-HPOD with a partially
purified fraction. Immediately after incubation, we
observed the production of both 3(Z)-nonenal (m=z ¼
122, 111, 96, 84, 69, 55, 41) and 2(E)-nonenal (m=z ¼
122, 111, 96, 83, 70, 55, 41), while after the reaction was
continued for 5 min, 3(Z)-nonenal disappeared and the
major product observed was 2(E)-nonenal. We found
neither 3(Z)-nonenal nor 2(E)-nonenal in the reaction
product from the boiled fraction with 9-HPOD. This
result indicates that the partially purified sample
contains both HPL and isomerization factor. The
isomerization factor was an enzyme, because the
partially purified fraction transformed 3(Z)-nonenal into
2(E)-nonenal, but it was inactivated by boiling
(Fig. 3B). Table 1 summarizes the results of partial
purification of 9-HPL-like activity. Following Resource
Q anion-exchange column chromatography, the specific
activity of 9-HPL-like activity increased 987-fold, and
the recovery rate was 1,025%. But these figures are
overestimated due to the presence of the inhibitory
Partial purification and characterization of 9-HPL-
like activity
Next we attempted to purify and characterize 9-HPL-
like activity. We did not use malt for enzyme purifica-
tion, because kilning (the heat-drying process for
germinating barley) causes modifications of malt pro-
teins, which disturb the structure analysis of the target
protein at the molecular level, such as N-terminal amino
acid sequence or LC–MS analysis. Instead, we used
embryos of germinating barley. A preliminary experi-

Characterization of 9-Fatty Acid Hydroperoxide Lyase-Like Activity
1665
Fig. 2. Partial Purification of 9-HPL-Like Activity from the Embryos of Germinating Barley Seeds by Anion Exchange Chromatography.
Desalted enzyme extract was applied to a Resource Q column equilibrated with 10 mM Tris–HCl (pH 7.2), 0.1% Tween20, and 1 mM ꢀ-
mercaptoethanol, and eluted by a NaCl gradient (0–1 M). Fractions of 0.5 ml were collected and assayed for 9-HPL-like activity. Symbols: open
circles, 9-HPL-like activity; solid line, concentration of NaCl (M); dotted line, absorbance at 280 nm.
Fig. 3. GC–MS Analysis of 9-HPL-Like Activity and 3Z:2E Isomerase.
A, Partially purified 9-HPL-like activity was mixed with 9-HPOD at a final concentration of 0.1 mM in a GC vial. After capping, a SPME fiber
was inserted either immediately (a), or after incubating at 30 ꢀC for 5 min (b), into the headspace of the vial. Aldehydes accumulated in a GC vial
were trapped with a SPME fiber for 5 min, and subjected to GC–MS analysis. Total ion chromatograms are presented. B, Partially purified 9-
HPL-like activity (test and boiled control) was incubated with 3(Z)-nonenal at 30 ꢀC for 20 min, and the products were extracted with ether
containing an internal standard (1-nonanol). A portion of ether layer was subjected to GC–MS analysis (SIM mode; select ion, m=z ¼ 69 and 70).
Table 1. Partial Purification of 9-HPL-Like Activity
9-HPL-like activity
3Z:2E Isomerase activity
Specific activity Recovery
Specific activity
(pkat/mg)
Recovery
(%)
Fractions
Crude extract
Gel filtration
Total protein (mg)
(pkat/mg)
(%)
26
24
0.3
0.63
296
100
194
11
11
100
92
Resource Q peak fraction
0.27
1025
180
18

1666
H. KURODA et al.
Table 2. Effect of Reagents on 9-HPL-Like Activity
Reagent
Concentration Relative activity (%)
100
None
Salicylic acid
Nordihydroguaiaretic acid
KCl
0.1 mM
1 mM
0.1 mM
1 mM
1 M
84
6.4
70
0
62
Fig. 4. Substrate–Velocity Plot of Partially Purified 9-HPL-Like
Activity.
The velocity of 9-HPL-like activity was measured at a concen-
tration of 0.0084–0.84mM. The apparent Km value was estimated to
be 23 mM.
factors. 3Z:2E Isomerase was co-purified with 9-HPL-
like activity. The specific activity was increased to 16-
fold, and the recovery rate was 18%. To characterize
HPL in 9-HPL-like activity, we further attempted
several purification methods to separate HPL and
3Z:2E isomerase, but these activities could not be
separated by chromatofocusing, cation-exchange, or
hydroxyapatite chromatography.
Fig. 5. Effect of pH on 9-HPL-Like Activity.
9-HPL-like activity was measured in either 0.1 M potassium
phosphate buffer (open circle, pH 5.0–8.0), 0.1 M MES–KOH (open
square, pH 5.5–6.5), or 0.1 M Tris–HCl (open triangle, pH 7.0–8.0).
Using this partially purified fraction which contained
both HPL and 3Z:2E isomerase activity, we performed
several biochemical analyses to characterize 9-HPL-like
activity. In every characterization, we monitored the
production of both 3(Z)-nonenal and 2(E)-nonenal, but
we observed only the production of 2(E)-nonenal.
First we examined the substrate-velocity plot (0.008–
0.84 mM) of 9-HPL-like activity (Fig. 4). The velocity of
9-HPL-like activity increased with a rise in substrate
concentration from 0 to 84 mM, while it decreased more
than 170 mM. At a concentration of 0.85 mM, activity
decreased to 11% of maximum, showing that 9-HPOD
acts as a suicide substrate. The apparent Km value for 9-
HPL-like activity was estimated to be 23 mM, similar to
that of HPL in dicots. The apparent Km values for 13-
HPOD were 14.6 mM with tea leaf HPL,17) and 20.8 and
22.1 mM with HPL I and II of bell pepper fruits.18)
Next, gel filtration chromatography was performed on
a portion of partially purified 9-HPL-like activity to
estimate its molecular weight (data not shown). Again,
we could not separate HPL and 3Z:2E isomerase. The
position of the peak active fraction coincided with the
eluting point of aldolase, showing that the molecular
weight of 9-HPL-like activity is approximately 160 kDa.
The molecular weight of bell pepper fruit fatty acid
hydroperoxide lyase was 170 kDa, consisting of a trimer
of 55 KDa polypeptide,18) and that of soybean HPL was
240–260 kDa.19)
9-HPL-like activity to 70% at a concentration of 0.1 mM,
and completely inhibited it at a concentration of 1 mM.
Salicylic acid reduced the activity to 84% and 6.4% at
concentrations of 0.1 mM and 1 mM respectively. In-
creasing the salt concentration (1 M KCl) resulted in
inhibition of 9-HPL-like activity to 62% (Table 2). The
pH-velocity plot of 9-HPL-like activity showed a typical
bellshape (pH 5–8), with maximum activity at pH 6.5 in
the potassium phosphate buffer (Fig. 5). Finally, we
examined the substrate specificity of partially purified
9-HPL-like activity by monitoring the decomposition
of hydroperoxide substrates. The relative activity toward
9-HPOD and 13-HPOD was 100:34.
Discussion
This paper characterizes 9-HPL-like activity in
relation to the flavor stability of beer, and shows the
biochemical properties of partially purified 9-HPL-like
activity from embryos of germinating barley seeds. We
found a positive correlation between 9-HPL-like activity
and the value of wort nonenal potential, an index for
predicting the staleness of stored beer. The staleness of
beer caused by lipid oxidation during mashing is often
described as a ‘‘cardboard flavor’’, and 2(E)-nonenal is
considered to be the major contributor of this flavor.3–5)
Next, we examined the effect of reagents and pH on
9-HPL-like activity. Nordihydroguiaretic acid reduced

Characterization of 9-Fatty Acid Hydroperoxide Lyase-Like Activity
1667
Recently, we discovered barley germplasms that lack
LOX-1, and the following brewing trial with malt made
from a malting barley crossed with one of these
germplasms revealed LOX-1 to have a significant effect
on the flavor stability of beer.20) Beer brewed with LOX-
less malt showed a higher sensory score with fewer
‘‘cardboard flavor’’ comments after storage compared
to the control line. Because LOX-1 produces only 9-
HPOD, we confirmed that the flavor compounds derived
from the decomposition or transformation of 9-HPOD
were the source of the cardboard flavor. The fact that
9-HPL-like activity cleaves 9-HPOD to produce 2(E)-
nonenal, and the fact that activity positively correlates
with the wort nonenal potential imply that not only
LOX-1, but also 9-HPL-like activity might be involved
in the formation of cardboard flavor.
Next, the possible involvement of 9-HPL-like activity
in beer flavor stability created the necessity of bio-
chemical and molecular analyses of this factor. Hence,
we attempted purification of this factor from the
embryos of germinating barley seeds. From the bio-
chemical analysis of partially purified 9-HPL-like
activity, it was shown that the activity consisted of
HPL and 3Z:2E isomerase (Fig. 6). Further attempts to
separate these activities failed, and hence we performed
several biochemical analyses using partially purified 9-
HPL-like activity which contained both HPL and 3Z:2E
isomerase. From these analyses, we obtained further
evidence that 9-HPL-like activity contained HPL. First,
9-HPL-like activity was inhibited by nordihydroguia-
retic and salicylic acids (Table 2). Because 3(Z)-nonenal
was not observed in all tests, it is thought that activity
was inhibited at the catalytic step of HPL. Lipophilic
antioxidants such as nordihydroguaiaretic acid are
thought to prevent the radical-formation step of the
catalytic activity of HPL,17) and salicylic acid is thought
to act as a metal-chelating reagent.21) Secondly, 9-HPL-
like activity was inhibited by a higher substrate
concentration (Fig. 4). HPL is thought to convert fatty
acid hydroperoxide into radical species, which in turn
destroy their own SH group, essential to catalytic
activity, thus inhibiting catalytic activity irreversibly.17)
At present, we do not have any information on HPL in
9-HPL-like activity as 9-/13-HPL. But, the presence of
HPL which cleaves 9-HPOD in germinating barley
seeds reminds one of the existence of both 13-HPL
(CYP74B) and 9-/13-HPL (CYP74C) in barley. The
HPL in 9-HPL-like activity characterized in this study
differed from barley 13-HPL14) on several points. First,
recombinant enzyme of barley 13-HPL expressed in
E. coli did not cleave 9-HPOD. Secondly, the pH-
velocity plots of 9-HPL-like activity and the barley
recombinant 13-HPL were different. Barley13-HPL had
slightly higher activity at pH 8.0 than at pH 7.0 in a
0.1 M potassium phosphate buffer, whereas the level of
9-HPL-like activity measured at pH 8.0 in the same
buffer was 44% of that measured at pH 7.0 (Fig. 5).
Thirdly, 9-HPL-like activity was inhibited by a higher
salt concentration, while barley 13-HPL was enhanced
(Table 2). In a previous report, we reported the presence
of 13-HPL-like activity that cleaved 13-HPOD into
hexanal, in addition to 9-HPL-like activity. These
activities were considered to be distinct because they
showed different sensitivity to proteinase-K.6) The
production of 2(E)-nonenal was reduced by proteinase-
K treatment, while hexanal production was not affected
by the same treatment. Assuming that 9-HPL-like
activity is 9-/13-HPL, part of the production of hexanal
would be reduced by proteinase-K treatment; however,
this effect would not be observed because the activity of
13-HPL-like activity was 100 times higher than that of
9-HPL-like activity. In our preliminary experiment, we
attempted to separate 9- and 13-HPL-like activities by
Resource Q anion exchange chromatography, which
directly indicates that these two activities are distinct
factors. However, we did not find a peak of 13-HPL-like
activity, presumably for the presence of other enzymes
which consume 13-HPOD. Whether 9- and 13-HPL-like
activities correspond to 9-/13-HPL and 13-HPL is to be
the subject of further work.
Although there are several reports showing the
relation between the quality of foods and the flavor
compounds derived from 13-HPOD or 13-HPOT by the
enzymatic action of 13-HPL or 9/13-HPL,11–13) the
effect of flavors developed from 9-HPOD by the action
of 9-/13-HPL on food quality is less fully character-
ized.6,22) This does not mean that the off-flavors derived
from 9-HPOD are less important, because as described,
2(E)-nonenal is thought to have a large influence on the
formation of the stale flavor of beer during storage.
Additionally, in rice, 2(E)-nonenal has been reported to
be a decisive element in determining the off-flavor of
Fig. 6. Pathway for the Production of 2(E)-Nonenal from 9-HPOD
with 9-HPL-Like Activity.
9-HPOD produced from the reaction of LOX-1 and linoleic acid
was cleaved into 3(Z)-nonenal with HPL. 3(Z)-Nonenal was further
isomerized into 2(E)-nonenal with 3Z:2E isomerase. 9-HPL-like
activity consisted of HPL and 3Z:2E isomerase.

1668
H. KURODA et al.
milled rice.23) It is intriguing that LOX3, the major LOX
found in the bran of rice seeds, specifically produces 9-
HPOD,24) as with barley LOX-1 in seeds or malts that
produce only 9-HPOD.15) We speculate that in cereals,
including barley and rice, the 9-LOX and 9-/13-HPL
cascade pathway that generates 2(E)-nonenal from
linoleic acid might have a significant effect on deteri-
oration in flavor quality.
Lipoxygenase and hydroperoxide lyase activities in
ripening strawberry fruits. J. Agric. Food Chem., 47,
249–253 (1999).
12) Nielsen, G. S., Larsen, L. M., and Poll, L., Formation of
aroma compounds during long-term frozen storage of
unblanched leek (Allium ampeloprasu Var. bulaga) as
affected by packaging atmosphere and slice thickness.
J. Agric. Food Chem., 52, 1234–1240 (2004).
´
13) Salas, J. J., and Sanchez, J., The decrease of virgin olive
oil flavor produced by high malaxation temperature is
due to inactivation of hydroperoxide lyase. J. Agric.
Food Chem., 47, 809–812 (1999).
Acknowledgments
14) Koeduka, T., Stumpe, M., Matsui, K., Kajiwara, T., and
Feussner, I., Kinetics of barley FA hydroperoxide lyase
are modulated by salts and detergents. Lipids, 38, 1167–
1172 (2003).
15) Kuroda, H., Kobayashi, N., Kaneda, H., Watari, J., and
Takashio, M., Characterization of factors that transform
linoleic acid into di- and trihydroxyoctadecenoic acids in
mash. J. Biosci. Bioeng., 93, 73–77 (2002).
We thank Toshiyuki Oshima and Koji Takazumi for
the GC–MS analysis. This work was partly supported by
a grant from Core Research for Evolutional Science and
Technology (Japan Science and Technology Agency).
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