Occurrence of bromoperoxidase in the marine green macro-alga, ulvella lens, and emission of volatile brominated methane by the enzyme

Article abstract of DOI:10.1016/S0031-9422(99)00404-5

Bromoperoxidase activity was detected in the marine green macro-alga, Ulvella lens, which is used to induce the larval metamorphosis of sea urchin in aquaculture in Japan. The enzyme activity was enhanced 8.5- and 2.2-fold by the addition of cobalt and vanadium ions to the reaction mixture, respectively. The volatile halogenated compounds dibromomethane and tribromomethane were formed in the reaction mixture when the enzyme was incubated with oxaloacetate, hydrogen peroxide and potassium bromide. These results suggest that dibromomethane, which was reported to be released by U. lens and play an important role as the inducer of larval settlement and metamorphosis, is produced by bromoperoxidase in the alga.

Full text of DOI:10.1016/S0031-9422(99)00404-5

Phytochemistry 52 (1999) 1211±1215
Occurrence of bromoperoxidase in the marine green macro-alga,
ulvella lens, and emission of volatile brominated methane by the
enzyme
a
Takashi Ohshiro , Satoru Nakano , Yoshinori Takahashi , Minoru Suzuki ,
b
b
b
b,
Yoshikazu Izumi *
a
Department of Biotechnology, Tottori University, Tottori 680-8552, Japan
Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan
b
Received 28 September 1998; received in revised form 5 May 1999
Abstract
Bromoperoxidase activity was detected in the marine green macro-alga, Ulvella lens, which is used to induce the larval
metamorphosis of sea urchin in aquaculture in Japan. The enzyme activity was enhanced 8.5- and 2.2-fold by the addition of
cobalt and vanadium ions to the reaction mixture, respectively. The volatile halogenated compounds dibromomethane and
tribromomethane were formed in the reaction mixture when the enzyme was incubated with oxaloacetate, hydrogen peroxide
and potassium bromide. These results suggest that dibromomethane, which was reported to be released by U. lens and play an
important role as the inducer of larval settlement and metamorphosis, is produced by bromoperoxidase in the alga. # 1999
Elsevier Science Ltd. All rights reserved.
Keywords: ulvella lens; Marine macro-alga; Green alga; Haloperoxidase; Bromoperoxidase; Volatile halogenated compounds; Sea urchin
1
. Introduction
bromoperoxidase (BPO) from C. pilulifera and expres-
sing it in Escherichia coli (Shimonishi et al., 1998).
Additionally, there are a few reports concerning halo-
peroxidases from green algae. A heme prosthetic group
was reported for enzymes from the green algae,
Penicillus capitatus (Manthey & Hager, 1981), P.
Many species of marine macro-algae contain a var-
iety of halogenated secondary metabolites (Niedleman
Geigert, 1986). A halogenating enzyme, haloperoxi-
&
dase (Niedleman & Geigert, 1986; Franssen, 1994), is
considered to participate in their syntheses in the pre-
sence of halides and hydrogen peroxide. Among them,
two bromoperoxidases from the red alga, Corallina
pilulifera (Itoh, Izumi & Yamada, 1985, 1986; Izumi,
lamourouxii (Barden
&
Corbett, 1980), and
Rhipocephalus phoenix (Barden & Corbett, 1980). It
was reported that BPO from the green alga, Halimeda
sp., was a non-heme enzyme (Butler & Walker, 1993),
but its detailed properties were not reported.
Ohshiro
& Wever, 1997) and the brown alga,
Ascophyllum nodosum (Wever, Plat & de Boer, 1985),
have been extensively studied, and were found to be
non-heme enzymes containing a unique prosthetic
group, vanadium (Krenn, Izumi, Yamada & Wever,
The biological halogenation by algal haloperoxidases
is considered to lead to the emission of the volatile
halogenated compounds such as CH I, CH Br, CH Cl
3
3
3
and CH Br2 (Wever, 1988; Wever, Tromp, Krenn,
2
Marjani
&
Abrahamsson & Pederse
van Toi, 1991; Colle
Â
Â
n, Ekdhal,
n, 1994; Itoh & Shinya, 1994;
1
1
989; de Boer, van Kooyl, Tromp, Plat & Wever,
986b). Recently we succeeded in cloning the gene of
Itoh, Tsujita, Ando, Hisatomi & Higashi, 1997), and
these compounds are recognized as substrates that
destroy the ozone layer. It has also been demonstrated
*
Corresponding author.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00404-5

1
212
T. Ohshiro et al. / Phytochemistry 52 (1999) 1211±1215
Table 1
Partial puri®cation of BPO from U. lens
Step
Total protein (mg)
Total activity (units)
Speci®c activity (units/mg)
Fold
Yield (%)
Cell-free extract
DEAE-Sepharose
Phenyl-Toyopearl
570
258
6.21
5.38
4.00
0.48
0.46
0.011
0.021
0.058
0.069
0.068
1
100
1.91
5.27
6.27
6.18
86.6
64.4
7.73
7.41
68.9
6.95
6.72
1
2
st Toyopearl HW-55
nd Toyopearl HW-55
that they are involved in the defense mechanism (alle-
lopathy) of the alga (Franssen, 1994). Such compounds
are one of several types of allelochemicals produced by
algae. For instance, the red alga, Neodilsea yendoana
produces a polyunsaturated fatty acid which inhibits
growth of the foliaceous green alga, Monostroma oxy-
spermum (Suzuki, Wakana, Denboh & Tatewaki,
mg-protein/ml) did not show any absorption peaks
other than at 280 nm; sodium azide did not inhibit
enzyme activity (data not shown), suggesting that the
enzyme did not have a heme prosthetic group. Various
metal ions were added to the reaction mixture to
examine their e€ects on BPO activity. As shown in
Table 2, 1 mM cobalt and vanadium ions enhanced
the enzyme activity 8.5- and 2.2-fold, respectively. On
1996), and C. pilulifera released volatile halomethanes
2+
2+
2+
which had suppressive e€ects on the development of a
brown alga, Laminaria angustata sporelings (Denboh,
Suzuki, Mizuno & Ichimura, 1997). In addition, a vol-
atile halogenated compound, dibromomethane, was
reported to induce larval settlement and metamorpho-
sis of the sea urchin, Strongylocentrotus nudus
the contrary, other metal ions, Ni , Zn , Cu
mM), and Fe
(1
(0.1 mM), inhibited the enzyme ac-
3+
tivity, suggesting that a metal might be involved in the
catalytic site. Although several vanadium-dependent
BPOs were found in marine macro-algae such as C.
pilulifera (Krenn et al., 1989), A. nodosum (de Boer et
al., 1986b), C. ocinalis (Sheeld, Harry, Smith &
Rogers, 1993) and Laminaria saccharina (de Boer,
Tromp, Plat, Krenn & Wever, 1986a), there are no
reports of cobalt-dependent haloperoxidases from mar-
ine macro-algae. It was reported that a bacterium,
Pseudomonas putida, produced BPO containing cobalt
ion (Itoh, Morinaga & Kouzai, 1994) and the metal
ion stimulated enzyme activity. The results obtained in
this study may indicate that BPO from U. lens is a
non-heme protein and cobalt-dependent. However,
further study is needed to examine the role of cobalt
and vanadium for BPO activity.
(
Taniguchi, Kurata, Maruzoi
&
Suzuki, 1994a;
Taniguchi, Kurata & Suzuki, 1994b). It was noted that
this inducer was released from several algae, including
the green alga, Ulvella lens.
The present study reports BPO activity in U. lens,
partial puri®cation of the enzyme from the green alga,
and the formation of volatile brominated compounds
by the partial puri®ed enzyme in the presence of oxa-
loacetate, hydrogen peroxide and potassium bromide.
2. Results and discussion
2.3. Volatile halide production from oxaloacetate by
BPO from U. lens
2.1. Partial puri®cation of bromoperoxidase
It was dicult to extract proteins from the green
Some organic acids, especially keto acids, are recog-
alga, U. lens; repeated operation of a Dyno-mill was
required to disrupt the cells, and led to the extraction
of only 570 mg protein from 500 g (wet weight) algae.
Further operation did not improve the extraction e-
ciency of protein from the algae. The speci®c activity
of U. lens enzyme in cell-free extract (0.011 units/mg
protein) was 30- and 90-fold less than those of C. pilu-
lifera (Itoh et al., 1985) and A. nodosum (Wever et al.,
Table 2
E€ect of metal ions on BPO activity
Compound
None
Conc. (mM)
Relative activity (%)
100
87
CaCl
MgSO
MnSO
CoSO
NiCl
ZnCl
CuCl
FeCl
NaVO
2
1
1
4
96
78
1985), respectively, which were reported to be high
4
1
producers of BPO. A typical partial puri®cation is
summarized in Table 1.
4
1
857
26
2
1
2
1
39
52
2
1
2.2. E€ect of metal ions on enzyme activity
3
0.1
1
30
221
3
The spectrum of the partially puri®ed enzyme (3.7

T. Ohshiro et al. / Phytochemistry 52 (1999) 1211±1215
1213
Fig. 1. GC-Mass spectra of the volatile compounds produced by BPO from U. lens. The mass spectra of the peaks at: (A) 13.8 min and (B) 19.3
min separated by GC.
nized as substrates for volatile halomethane formation
by BPO. It was shown that a marine red alga,
Bonnemaisonia hamifera, produced bromoform and
dibromomethane from 3-oxooctanoic acid (Theiler,
Cook & Hager, 1978). The simpler keto acids includ-
ing oxaloacetate and 2-ketoglutarate were shown to
serve as the substrate of the puri®ed BPO from C. pilu-
lifera, and bromomethane was released (Itoh &
Shinya, 1994). In the present report, the formation of
volatile halomethanes was examined using the partially
puri®ed BPO preparation from U. lens according to
the method used for the C. pilulifera enzyme (Itoh &
Shinya, 1994). When the enzyme reaction was per-
formed with oxaloacetate as a halogen acceptor, the
GC chromatogram of volatile products showed new
peaks (retention time; 13.8 and 19.3 min) which did
not appear in control lacking BPO. The GC-MS spec-
tra of the new peaks are shown in Fig. 1. The peaks
with retention time at 13.8 and 19.3 min corresponded
to dibromomethane and tribromomethane, respect-
ively. In this experiment, the enzyme from U. lens cata-
lyzed the halogenation reaction without the presence
of added cobalt or vanadium in the reaction mixture.
As shown in Table 2, the enzyme from U. lens was
active without added cobalt or vanadate. In the case
of C. pilulifera, the puri®ed, vanadium-dependent BPO
was active without adding vanadate to the reaction
mixture (Itoh et al., 1985; Krenn et al., 1989). The
enzymes of these algae may contain some metal(s)
required for activity, which are not removed by the
puri®cation procedures.
Thus, these data demonstrate that BPO from U. lens
could produce volatile halomethane from oxaloacetate;
volatile halomethane was previously reported to induce
larval settlement and metamorphosis of sea urchin by
Taniguchi et al. (1994a, 1994b).

1
214
T. Ohshiro et al. / Phytochemistry 52 (1999) 1211±1215
3. Experimental
The enzymatic reaction to release the volatile halo-
genated compounds was carried out according to the
procedure described previously (Itoh & Shinya, 1994).
The reaction mixture contained 0.1 M potassium phos-
phate bu€er (pH 6.8), 10 mM KBr, 1 mM oxaloacetic
acid, 1 mM H O and 0.0023 units/ml BPO from U.
lens in a total volume of 30 ml. A vial (30 ml head-
space) containing the reaction mixture was allowed to
stand for 2 h at 208C. A control was performed with-
out BPO. The gas phase was analyzed by the head-
space method using a GC-MS instrument. Analysis of
the products was performed with a Hewlett Packard
gas chromatograph (HP5890-II) ®tted with a capillary
column (Rtx-volatiles, 60 m: 0.25 mm id: 1 mm) and a
Hewlett Packard mass spectrometer (HP-5971A).
Ulvella lens was grown on transparent, colorless
acrylic resin plates (580 Â 300 Â 0.5 mm) for approxi-
mately two months at the Hokkaido Aquaculture
Development Authority, Kayabe-gun, Hokkaido, in
early spring, then scraped by spatulas and stored at
208C until use. Enzyme puri®cation was performed
below 158C. Algal cells (500 g, wet weight) were sus-
2
2
�
pended in 500 ml of 50 mM Tris-SO bu€er (pH 7.4)
4
and disrupted by f 0.5 mm glass beads through a
Dyno-mill homogenizer (Willy A. Bachofen, Basel,
Switzerland) ®ve times. The cell-free extract was
obtained by centrifugation at 12,000 Â g for 30 min,
and dialyzed against 50 mM Tris-SO bu€er (pH 7.4).
4
The dialyzed enzyme solution was applied onto a
DEAE-Sepharose Fast Flow column ꢀf 3.6 Â 30 cm)
equilibrated with 50 mM Tris-SO bu€er (pH 7.4). The
4
Acknowledgements
column was washed with the same bu€er, and the
enzyme was eluted with the same bu€er containing 0.8
M NaCl at a ¯ow rate of 60 ml/h. The active fractions
We thank the Hokkaido Aquaculture Development
Authority for cultivation and supply of U. lens cells,
and Dr. R. Wever, University of Amsterdam, for
instruction in the phenol red assay method of BPO ac-
tivity. We are also grateful to Dr. Hideharu Kondoh,
Hokkaido Institute of Environmental Science, for use
of the GC-MS instrument. Part of this work was ®nan-
cially supported by a Grant-in Aid for Scienti®c
Research from the Ministry of Education, Science,
Sports and Culture of Japan.
were combined, dialyzed against 50 mM Tris-SO buf-
4
fer (pH 7.4) containing 0.8 M (NH ) SO , and applied
4
2
4
onto a Phenyl Toyopearl column ꢀf 1.8 Â 19 cm) equi-
librated with the dialysis bu€er. The column was
washed with the same bu€er and eluted with a linear
(
SO bu€er (pH 7.4), using a ¯ow rate of 10 ml/h. The
NH ) SO gradient (from 0.8 to 0 M) in 50 mM Tris-
4 2 4
4
active fractions were combined and concentrated by
ultra®ltration. The concentrated enzyme was applied
to a Toyopearl HW-55 gel ®ltration column ꢀf
3
.6  30 cm) equilibrated with 50 mM Tris-SO bu€er
4
(
pH 7.4) containing 0.1 M KCl at a ¯ow rate of 3 ml/
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