▪ Abstract Alginate lyases, characterized as either mannuronate (EC 4223) or guluronate lyases (EC 42211), catalyze the degradation of alginate, a complex copolymer of α-L-guluronate and its C5 epimer β-D-mannuronate Lyases have been isolated from a wide range of organisms, including algae, marine invertebrates, and marine and terrestrial microorganisms This review catalogs the major characteristics of these lyases, the methods for analyzing these enzymes, as well as their biological roles Analysis of primary sequence data identifies some markedly conserved motifs that should help elucidate functional domains Information about the three-dimensional structure of a mannuronate lyase from Sphingomonas sp, combined with various mutagenesis studies, has identified residues that are important for catalytic activity in several lyases Characterization of alginate lyases will enhance and expand the use of these enzymes to engineer novel alginate polymers for applications in various industrial, agricult

ALGINATE LYASE: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications.

Enzymic degradation of alginates

The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Various abalones and sea-hares from California and Brittany (France) were surveyed for their cellulase, fucanase, protease and alginate lyase activities. The crude digestive juices contained significant amounts of cellulase, fucanase and alginate lyase but very low levels of protease. The alginate lyases from both the sea hare Aplysia depilans and the abalone Haliotis tuberculata preferentially degraded polymannuronate sequences. Haliotis tuberculata alginate lyases were partially purified by cation exchange chromatography. For the purpose of protoplast preparation from brown algae, the purification procedure was scaled up to produce about 30 000 units of alginate lyase activity, with a 100-fold purification coefficient, in one run. This fraction did not retain cellulase, fucanase or proteolytic activities.

Preparation of alginate lyases from marine molluscs for protoplast isolation in brown algae

SUMMARY
Advances have been made in cell and tissue culture of seaweeds to define a unique branch of in vitro techniques; however, they are lagging far behind those of land plants and have limited applications. Explants can be cultivated axenically in enriched or artificial seawater culture media, and regeneration and even callus formation are achieved. In this state of the art technique, seaweed tissue culture may be already useful for certain biotechnological applications, such as clonal propagation of seed material for mariculture. Nevertheless, the absolute control of growth and development as it is exerted in higher plant tissue culture is lacking, and it is required for more complex biotechnological applications in seaweeds. Definitively, we need appropriate cells (competent cells) to induce growth with the most effective chemical regulators in culture medium adjusted towards the addition of carbon sources. Still, free cells and protoplast isolation and regeneration in marine seaweeds constitute the most developed topic in seaweed tissue culture. The regulation of growth and development of seaweed free cell and protoplast cultures may sustain a purposeful use of techniques in the era of genomic applications.

Review: Seaweed tissue culture as applied to biotechnology: Problems, achievements and prospects

Protoplast were isolated from a filamentous brown alga, Sphacelaria sp. (Sphacelariales, Phaeophyta), using alginate-lyases extracted from marine molluscs, and commercial pectinase and cellulase. Yields were about 4000 protoplasts per gram of fresh tissue. Different types of protoplasts, originating from apical, subapical, nodal and internodal cells, could be readily identified based on their size and pigmentation. Apical cells produced a higher percentage of protoplasts (approx. 2%), compared with other cell types. All apical-cell protoplasts regenerated into new thalli and most other types of protoplasts divided at least once in culture, but did not develop further.

G. Ducreux

Papers

Plant regeneration from protoplasts of Sphacelaria (Phaeophyceae)