The polysaccharides are polymers and they constitute the essential elements of the green algae of the genus Ulva. Moreover, these green algae are very widely distributed on the coasts of all the continents and consequently are widely available at low cost.
These polymers are called “ulvan” and they contain acidic monosaccharides, notably glucuronic acid (GlcpA) and iduronic acid (IdopA) as well as neutral monosaccharides including rhamnose (Rhap), galactose (Galp), glucose (Glcp) and xylose (Xylp). Moreover, in these polymers, the rhamnose is substituted with a sulfate group on carbon 3, which will be called rhamnose 3 sulfate (Rhap3S).
These polymers contain interesting compounds, in particular iduronic acid and rhamnose 3 sulfate, notably for therapeutic or cosmetic applications.
However, the monosaccharides of these polymers are linked together relatively randomly, so that it is difficult to extract a relatively homogeneous composition. However, two main sequences, called aldobiuronic acids, including said compounds of interest, have been identified, a first sequence [→4)-β-D-GlcpA-(1→4)-α-L-Rhap3 sulfate-(1→]n and a second sequence [→4)-α-L-IdopA-(1→4)-α-L-Rhap3 sulfate-(1→]m. Sequences containing glucuronic acid chains, i.e.: [→4)-β-D-GlcpA-(1→4)-β-D-GlcpA-(1→]x have also been described; these glucuronan sequences can be integrated in the 2 main sequences constituted of the aforementioned aldobiuronic acids. However, although it has not been proved, these glucuronan sequences might also be present on a polymer other than ulvan, but extracted from the alga at the same time as the polymer constituted predominantly of sequences of aldobiuronic acids.
These compounds are comparable, respectively, to the glycosaminoglycans, notably heparin and heparan sulfate, and to chondroitin sulfate and dermatan sulfate.
However, as interesting as they might be, these polymers extracted from green algae are too heterogeneous to be used and incorporated as they are in pharmaceutical or cosmetic preparations. Accordingly, their fractionation into elementary compounds by means of specific enzyme compositions has been considered.
Thus, a first enzyme in the class of the lyases, glucuronan lyase, has already been identified, and it is active in cleaving the glucuronan sequences present in polysaccharides of the ulvan type. Moreover, this enzyme is obtained from a fungal microorganism, in particular from the strain Trichoderma CNCM I-3400. Reference may be made to document FR 2 885 911, which describes this microorganism for breaking down the glucuronan sequences of polysaccharides.
However, this enzyme composition does not actually permit cleavage of the aforementioned main sequences constituted of aldobiuronic acids, the result of which would be of interest.
Nevertheless, another enzyme composition in the class of the lyases is known, ulvan lyase, which makes it possible, a priori, to cleave the first and second sequences of the aforementioned aldobiuronic acids at the glycoside bonds, between units Rhap3 sulfate and GlcpA on the one hand, and between units Rhap3 and IdopA on the other hand, and according to a reaction of β elimination.
This enzyme composition is described in the work of M. Lahaye et al. (Carbohydrate Research 1997, 304: 325-333) and it is derived from a marine bacterium. It makes it possible to fractionate the polymers of ulvan into oligomers.
However, the enzyme composition described in that work and notably its enzymatic activity relative to the substrate comprising ulvan polymers is relatively weak, and furthermore, it decreases rapidly as degradation proceeds, so that it is necessary to reintroduce enzymes of said enzyme composition for degradation to continue. In fact, the work cited above states that the degradation of ulvan by the isolated enzyme extract is slow and quickly reaches a plateau. Thus, it is described there that the increase in reducing sugars on ulvan submitted to degradation by the enzyme, said increase logically being identical to that of the sugars in the nonreducing terminal position, is 7.1% relative to the content of these sugars before incubation in the presence of the enzyme; furthermore, this degradation obtained in 200 minutes requires repeated additions of enzyme, on average every 25 minutes, to compensate the inactivation of the enzyme by degradation products. Consequently, use of this enzyme composition cannot be considered for industrial production of oligomers with uniform compositions and molecular weight, as the cost of obtaining these oligomers would be prohibitive.
Thus, a problem that arises, and that the present invention aims to solve, is to provide, according to a first aspect, a method of enzymatic cleavage that would make it possible not only to cleave the ulvan polymers between units Rhap3 sulfate and GlcpA, and between units Rhap3 and IdopA, but that would also make it possible to do so with a productivity enabling the oligomers of reduced size to be obtained cost-effectively.