Ricardo Melo

Vanadium haloperoxidases from brown algae of the Laminariaceae family.

Vanadium haloperoxidases were extracted, purified and characterized from three different species of Laminariaceae--Laminaria saccharina (Linné) Lamouroux, Laminaria hyperborea (Gunner) Foslie and Laminaria ochroleuca de la Pylaie. Two different forms of the vanadium haloperoxidases were purified from L. saccharina and L. hyperborea and one form from L. ochroleuca species. Reconstitution experiments in the presence of several metal ions showed that only vanadium(V) completely restored the enzymes activity. The stability of some enzymes in mixtures of buffer solution and several organic solvents such as acetone, ethanol, methanol and 1-propanol was noteworthy; for instance, after 30 days at least 40% of the initial activity for some isoforms remained in mixtures of 3:1 buffer solution/organic solvent. The enzymes were also moderately thermostable, keeping full activity up to 40 degrees C. Some preliminary steady-state kinetic studies were performed and apparent Michaelis-Menten kinetic parameters were determined for the substrates iodide and hydrogen peroxide. Histochemical studies were also performed in fresh tissue sections from stipe and blade of L. hyperborea and L. saccharina, showing that haloperoxidase activity was concentrated in the external cortex near the cuticle, although some activity was also observed in the inner cortical region.

Light response curves for Gelidium sesquipedale from different depths, determined by two methods: O2 evolution and chlorophyll fluorescence

Photosynthesis-light response curves of Gelidium sesquipedale from the west coast of Portugal (Cape Espichel) were determined at four different depths, 3, 10, 15 and 22 m. Data acquisition using chlorophyll a fluorescence methodology and oxygen electrode measurements were compared. Response curves were determined over an increasing range of irradiance values (I), from darkness to 900 μmol photon m-2 s-1 PAR. In general, light response curves obtained for G. sesquipedale showed a similar pattern whether determined by the chlorophyll fluorescence method or by oxygen evolution. The photosynthetic capacity of G. sesquipedale decreased with depth, as expected, revealing a ‘sun’ and ‘shade’ acclimation pattern, between shallow and deeper waters.

Saccorhiza polyschides (phaeophyceae; phyllariaceae) A new source for vanadium-dependent haloperoxidases

Abstract Vanadium-dependent iodoperoxidases from the brown seaweed Saccorhiza polyschides (Lightfoot) Batters, collected at three different locations along the Portuguese west coast, were extracted, purified and characterized. Several extraction procedures were tested, including two-phase aqueous systems. The purification of the iodoperoxidases was achieved using hydrophobic interaction chromatography followed by chromatofocusing. It was possible to isolate three different isoforms of the enzyme, which show mainly iodoperoxidase activity. The three native enzymes have a relative M r around 125 kDa, and two subunits of M r about 64 kDa. Reactivation studies of the apoenzymes with several metal ions revealed that vanadium(V) was essential for enzymatic activity. These enzymes are remarkably thermostable, maintaining their maximum activity up to 50°. The kinetic parameters for the enzyme catalysed iodoperoxidase reaction were obtained at pH 6.1. In the concentration range studied (0.2–8 mM) there was no inhibition by H 2 O 2 whereas iodide inhibition was already apparent at the top values of the concentration range studied (2–25 mM).

Gelidium commercial exploitation: natural resources and cultivation

The state of the Gelidium agarophyte resource was last reviewed in 1990 during the First International Workshop on Gelidium. The main objective of the present study was to gather and analyse the new information made available since then, with emphasis on the G. sesquipedale resource from the Iberian Peninsula and Morocco. Other Gelidium-based resources world-wide are also reviewed, as well as new attempts to cultivate Gelidium species in the sea. In general, since the late 1980s, G. sesquipedale yields have decreased in Portugal, and increased in Spain and Morocco. Current reported values, in dry weights, are ca. 670 t yr-1 for Portugal (average 1995–1997), 5200 t yr-1 in Spain, and 6950 t yr-1 for Morocco (average 1994–1996). Efforts to develop cultivation of G. sesquipedale in the sea in Spain have yet to progress beyond the demonstration scale. Other important Gelidium-based agarophyte resources are located in: (i) South Africa where, in addition to G. pristoides, new species are being harvested, with total agarophyte landings of 140 t yr-1 in 1996; (ii) Mexico, where G. robustum harvest averaged 750 t yr-1 in 1987–1996; (iii) and Chile where harvest of Gelidium species yielded ca. 460 t yr-1 in 1990–1992. Preliminary research on new techniques for commercial cultivation of Gelidium species is also reported from these countries.

Purification and characterisation of vanadium haloperoxidases from the brown alga Pelvetia canaliculata

Two enzymes characterised as iodoperoxidases (PcI and PcII), with vanadium-dependent activity, have been purified from the brown alga Pelvetia canaliculata (L.) Decne et Thur. (Fucaceae, Phaeophyceae), collected in the Northern Portuguese coast, at Viana do Castelo. The relative molecular masses were 166 kDa for PcI and 416 kDa for PcII, as determined by gel filtration. SDS-PAGE shows that PcI has just one band corresponding to a subunit of 66 kDa, while PcII shows four bands (66, 72, 157 and 280 kDa). The following kinetic parameters have been determined from a steady-state analysis of the oxidation of iodide by H2O2: PcI, pHopt = 6.0, KM(I-) = 2.1 mM, KM(H2O2) = 110 microM, Ki(I-) = 127 mM; and PcII, pHopt = 6.5, KM(I-) = 2.4 mM, KM(H2O2) = 20 microM and Ki(I-) = 69 mM. These iodoperoxidases are thermostable, as also observed for vanadium bromo- and chloroperoxidases.

Life history and reproductive potential of the agarophyte Gelidium robustum in California

The reproductive potential of the tetrasporangial phase of Gelidium robustum was studied for 16 months at two sites off Santa Barbara, California. In all samples tetrasporangial thalli were always more abundant than gametangial ones. Tetratrasporangial sori were present throughout the duration of the study but relative fecundity was highest [300–400 sori g−1 (w. wt)] in spring/summer samples of consecutive years, as a result of increasing numbers both of tetrasporangial branchlets per plant and of sori per branchlet. On the other hand, laboratory experiments showed that tetraspore release per sorus was highest (150–250 spores sorus−1 d−1) in winter. Inferring from these field and laboratory data plants released up to ± 34 000 tetraspores g−1 (w. wt) d−1 in the spring/summer of the second study year. Tetraspore germination, under defined culture conditions, also showed a marked seasonality increasing sharply from less than 10% in winter up to almost 60% in spring/summer, thus coinciding with the period of maximal spore output per plant. These results suggest that although relatively high numbers of tetraspores may be released by G. robustum plants all year round these might not always have the potential to germinate and recruit.

Gelidium cultivation in the sea

Gelidium fronds were grown in the sea under a variety of experimental conditions: on rigid, damped and tensioned test farms of various designs, in calmer and more turbulent habitats, at various depths, with and without commercial fertilizer supply. Initially, the effectiveness of a given cultivation strategy was based on the survival and growth of the fronds, here termed ‘bio-assay’ mariculture. Ambient seawater temperature, nutrient availability, hydrodynamics and other environmental parameters were measured periodically. In-the-sea irrigation of test plants with commercial fertilizers was apparently effective, at least with some farm designs, and when ambient nutrient levels were low. Under optimal conditions, achieved through experimental manipulation of farm design, specific growth rates of over 2% per day were recorded. However, considerable variation in growth rates and in plant performances was observed. It was not always possible to correlate these variations with design modifications or other experimental parameters. In view of these findings, we have reviewed our initial ‘bio-assay’ approach, namely the assumptions about the design and operation of farm structures and their interactions with the water and the fronds. Methods were developed to quantify these interactions. We advocate a quantitative, ‘hydrodynamic’ approach in developing an effective cultivation strategy for gelidioid algae and are optimistic about progressing from test-to-commercial scale farms in the near future.