*3.6. Enzymatic Degradation*

The HA/Ch enzymatic degradation with lysozyme and hyaluronidase was studied. Such tests provide information on the polysaccharide stability in a physiological environ-

ment. The samples incubated at different times were analyzed. The Mw values vs. time, determined by chromatographic elaboration, are shown in Figure 8.

**Figure 8.** (**a**) HA and (**b**) Ch kinetic degradation with HAse (green curves), Lys (black curves), and a mix of enzymes (red curves) over 24 h.

η The HA treatment (Figure 8a, green line) resulted in a substantial reduction of Mw indicating depolymerization, and such depolymerization took place very quickly during the initial 3 h, this indicated that the enzymatic hydrolysis was an endo-action. After 3 h, degradation slowed down, probably due to the inhibition of the enzyme activity by the end products, and the reaction was completed after 24 h, at the end of which a reduction of the Mw of 98% was observed (from 992 kDa to 14 kDa). In the case of Lys (Figure 8a, black line), the enzyme did not have any degradation effect, and Mw remained constant for the incubation time (24 h). As expected, the presence of Lys in complex with HAse (Figure 8a, red line) had a mild effect on the HA hydrolysis, showing a slightly different initial degradation rate, but the end point was the same.

**η / η / η** For Ch, in the enzymatic degradation with Lys (Figure 8b, black line), the Mw decreased slowly and, after 24 h of incubation, the value was reduced by 86% (from 970 kDa to 137 kDa); with HAse (Figure 8b, green line) a small reduction of Mw was observed, at about 20%, which was in agreement with the lack of a specific substrate for this enzyme. The decrease of Mw was the same with the complex of enzymes than with Lys alone as expected, the "driving force" for the degradation of Ch was Lys but the presence of hyaluronidase seemed to induce an effect on the first part of hydrolysis process.

To study how the interactions between HA and Ch could affect the enzymatic activity, the complex was hydrolyzed. The results with HAse and Lys individually and finally with enzymatic complex are shown in Table 6. All chromatograms of solutions until 6 h of incubation present only one peak, derived from HA and Ch, due to the very similar molecular weight. Consequently, the Mw and [η] values are related to the complex, imposing the *dn*/*dc* of 0.118 (see HP-SEC-TDA results section).

η After 6 h, the Mw was reduced by about 70% with HAse, and 20% and 92% with Lys and with HAse + Lys, respectively. Therefore, the enzyme activity was not affected by the combination of the two polysaccharides.

After 24 h of incubation, the chromatograms present two distinct peaks (Figure 9), one related to the HA component and the other one attributed to Ch, so Mw and [η] for each polysaccharide could be determined, using the specific *dn*/*dc* values. The results are presented in Table 7.


**Table 6.** HP-SEC-TDA results of the HA/Ch complex degradation.

**Figure 9.** RI signal from the HP-SEC-TDA analysis of the HA/Ch complex at t = 0 (black) and after degradation t = 24 h with HAse (red), Lys (blue), and a mix of enzymes (green).


**Table 7.** HP-SEC-TDA results of the HA/Ch complex degradation after 24 h.

The Mw values were very similar to the results obtained for the solution aliquots containing only HA or Ch, at 24 h, suggesting that the co-presence of both polysaccharides did not affect the enzymatic action.
