Dispersed cellulose ribbons from bacterial cellulose were subjected to digestion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an excess of ␤-glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than Cel6A. In combination, these enzymes showed substantial synergy culminating with a molar ratio of approximately two-thirds Cel6A and one-third Cel7A. Ultrastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations, together with the examination of the digestion kinetics, indicate that Cel6A can be classified as an endo-processive enzyme, whereas Cel7A is essentially a processive enzyme. Thus, the synergy resulting from the mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an appropriate substrate, superior to Valonia or bacterial cellulose microcrystals, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A.Despite a large number of studies, the mechanism of the enzymatic digestion of crystalline cellulose stands as a major unsolved problem of persisting industrial and scientific significance. As early as 1950, it was realized that the degradation of cellulose required a complex of enzymes working together (36). This crucial observation has been confirmed by an extensive number of studies. Following these reports, a general picture has emerged indicating that at least three types of enzymes need to cooperate to digest efficiently crystalline cellulose into glucose: (i) endoglucanases (EC 3.2.1.4), which cut the cellulose chains randomly; (ii) cellobiohydrolases (CBH) (EC 3.2.1.91), which recurrently cleave cellobiose from the cellulose chain ends; and (iii) ␤-glucosidases (EC 3.2.2.21), which hydrolyze cellobiose and various soluble cellodextrins into glucose (reviewed in references 4, 15, 16, 22, 30, 42, 44, and 53).The complementary activities of the different enzymes is thought to be responsible for synergistic effects, whereby the enzymatic activity of a mixture of two or several enzymes is substantially higher than the sum of the activities of the individual enzymes. Several types of synergy have been described, the easier to apprehend being the cooperation action of endoand exo-acting enzymes on cellulose (21,28,35,49,50). In such a cooperation, the action of endocellulases is to increase the number of chain ends and, therefore, to enhance the action of exocellulases, which themselves appear to be the key enzymes for the digestion of crystalline cellulose. In this context, one of the main characteristics of CBH is that they act on cellulose chains in a "processive" manner (10,23,27,37,38,45), as they progress along the polymer chain while releasing cello...

Alginate is an industrially important polysaccharide obtained commercially by harvesting brown algae. The final step in alginate biosynthesis, the epimerization of ␤-1,4-d-mannuronic acid to ␣-1,4-l-guluronic acid, a structural change that controls the physicochemical properties of the alginate, is catalyzed by the enzyme mannuronan C-5-epimerase. Six different cDNAs with homology to bacterial mannuronan C-5-epimerases were isolated from the brown alga Laminaria digitata (Phaeophyceae). Hydrophobic cluster analysis indicated that the proteins encoded by the L. digitata sequences have important structural similarities to the bacterial mannuronan C-5-epimerases, including conservation of the catalytic site. The expression of the C-5-epimerase genes was examined by northern-blot analysis and reverse transcriptase-polymerase chain reaction in L. digitata throughout a year. Expression was also monitored in protoplast cultures by northern and western blot, reverse transcriptase-polymerase chain reaction, and activity measurements. From both the structural comparisons and the expression pattern, it appears that the cDNAs isolated from L. digitata encode functional mannuronan C-5-epimerases. The phylogenetic relationships of the bacterial and brown algal enzymes and the inferences on the origin of alginate biosynthetic machinery are discussed.Kelps are photosynthetic organisms that are abundant on rocky coasts from temperate to sub-Antarctic and Arctic seas. They are members of the class Phaeophyceae in the division Heterokontophyta, a phylum that emerged as an independent lineage contemporarily with the radiation of other higher eukaryote lineages (Baldauf et al., 2000). They have a haplodiplophasic life cycle, alternating between a microscopic gametophyte phase and a macroscopic sporophyte phase. Although the gametophyte phase consists of uniseriate filaments, a few millimeters long, the sporophytes have distinct organs, i.e. holdfast, stipe, and blade, and can reach from several to 50 m in length depending on the species. The kelp sporophytes are an economically important marine crop, cultivated in East Asia and harvested from natural populations in Europe and North America, including for the production of alginate, a gelforming polysaccharide used in a large variety of pharmaceutical, food, and industrial applications (Onsøyen, 1996).Alginate is the major matrix component of brown algal cell walls, up to 45% in dry weight (Kloareg and Quatrano, 1988). It consists of an unbranched polysaccharide made of ␤-1,4-d-mannuronic acid (M) and its C-5-epimer ␣-1,4-l-guluronic acid (G), which are arranged in homopolymeric regions of M and G blocks, interspaced with regions of alternating structure (MG blocks). These features vary according to the nature and the age of the tissues as well as to the season and to the growing area (sheltered or exposed). The stipe of Laminaria spp., for example, contains a high fraction of G blocks, whereas the blade contains a higher proportion of M residues (Haug et al., 1974). The biological...