Elsevier

Phytochemistry

Volume 52, Issue 7, December 1999, Pages 1211-1215
Phytochemistry

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

https://doi.org/10.1016/S0031-9422(99)00404-5Get rights and content

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.

Introduction

Many species of marine macro-algae contain a variety of halogenated secondary metabolites (Niedleman & Geigert, 1986). A halogenating enzyme, haloperoxidase (Niedleman & Geigert, 1986; Franssen, 1994), is considered to participate in their syntheses in the presence of halides and hydrogen peroxide. Among them, two bromoperoxidases from the red alga, Corallina pilulifera (Itoh et al., 1985, Itoh, Izumi & Yamada, 1986, Izumi, 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 et al., 1989, de Boer et al., 1986b). Recently we succeeded in cloning the gene of bromoperoxidase (BPO) from C. pilulifera and expressing it in Escherichia coli (Shimonishi et al., 1998). Additionally, there are a few reports concerning haloperoxidases from green algae. A heme prosthetic group was reported for enzymes from the green algae, Penicillus capitatus (Manthey & Hager, 1981), P. 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.

The biological halogenation by algal haloperoxidases is considered to lead to the emission of the volatile halogenated compounds such as CH3I, CH3Br, CH3Cl and CH2Br2 (Wever, 1988; Wever, Tromp, Krenn, Marjani & van Toi, 1991; Collén, Ekdhal, Abrahamsson & Pedersén, 1994; Itoh & Shinya, 1994; Itoh, Tsujita, Ando, Hisatomi & Higashi, 1997), and these compounds are recognized as substrates that destroy the ozone layer. It has also been demonstrated that they are involved in the defense mechanism (allelopathy) 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 oxyspermum (Suzuki, Wakana, Denboh & Tatewaki, 1996), and C. pilulifera released volatile halomethanes which had suppressive effects on the development of a brown alga, Laminaria angustata sporelings (Denboh, Suzuki, Mizuno & Ichimura, 1997). In addition, a volatile halogenated compound, dibromomethane, was reported to induce larval settlement and metamorphosis of the sea urchin, Strongylocentrotus nudus (Taniguchi et al., 1994a, Taniguchi et al., 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 purification of the enzyme from the green alga, and the formation of volatile brominated compounds by the partial purified enzyme in the presence of oxaloacetate, hydrogen peroxide and potassium bromide.

Section snippets

Partial purification of bromoperoxidase

It was difficult to extract proteins from the green 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 efficiency of protein from the algae. The specific activity of U. lens enzyme in cell-free extract (0.011 units/mg protein) was 30- and 90-fold less than those of C. pilulifera (Itoh et al., 1985) and A. nodosum (Wever et al., 1985),

Experimental

Ulvella lens was grown on transparent, colorless acrylic resin plates (580 × 300 × 0.5 mm) for approximately two months at the Hokkaido Aquaculture Development Authority, Kayabe-gun, Hokkaido, in early spring, then scraped by spatulas and stored at −20°C until use. Enzyme purification was performed below 15°C. Algal cells (500 g, wet weight) were suspended in 500 ml of 50 mM Tris-SO4 buffer (pH 7.4) and disrupted by φ 0.5 mm glass beads through a Dyno-mill homogenizer (Willy A. Bachofen, Basel,

Acknowledgments

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 activity. 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 financially supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

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