*3.5. Enzymatic Susceptibility of Chondbiuronan*

GAG can be enzymatically depolymerized either by eliminative cleavage with lyases (EC 4.2.2.-) resulting in disaccharides or oligosaccharides with ∆4,5-unsaturated uronic acid residues at their non-reducing end or by hydrolytic cleavage with hydrolases (EC 3.2.1.-).

Chondroitin AC lyase from *Flavobacterium heparinum* (EC 4.2.2.5) cleaves a variety of GAGs, including chondroitin sulfates A (chondroitin 4-sulfate) and C (chondroitin 6-sulfate), as well as the unsulfated chondroitin and hyaluronic acid [22]. Bovine testis hyaluronidase (EC 3.2.1.35) is an endo-β-N-acetyl-D-hexosaminidase that hydrolyses hyaluronan and to a lesser extent sulfated or unsulfated chondroitin [23].

Here, the capacity of chondbiuronan to be degraded by these enzymes was evaluated compared to unsulfated chondroitin. As shown in Figure 6, both enzymes could degrade chondbiuronan. Hydrolysis rates of chondbiuronan by chondroitin AC lyase and hyaluronidase were however decreased of about 10-fold and 30-fold respectively, when compared to chondroitin.

**Figure 6.** Enzymatic digestion of chondbiuronan and chondroitin by chondroitin AC lyase (**A**) and by hyaluronidase (**B**). The filled or hollow circles represent activity on chondroitin and chondbiuronan respectively. A<sup>235</sup> is proportional to the concentration of unsaturated non reducing ends formed by the lyase action. Hyaluronidase activity was followed by measuring reducing sugars released from substrates with the ferricyanide method.
