Laccase-Based CLEAs: Chitosan as a Novel Cross-Linking Agent

Laccase from Coriolopsis Polyzona was insolubilized as cross-linkedenzyme aggregates (CLEAs) for the first time with chitosan as the cross-linkingagent. Concentrations between 0.01 and 1.867 g/L of chitosan were used andbetween 0.05 and 600 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride. The laccase was precipitated using ammonium sulphate and cross-linkedsimultaneously. Specific activity and thermal stability of these biocatalysts weremeasured. Activities of up to 737 U/g were obtained when2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used as asubstrate. Moreover, the stability of these biocatalysts was improved with regardsto thermal degradation compared to free laccase when exposed to denaturingconditions of high temperature and low pH. The CLEAs stability against chemicaldenaturants was also tested but no significant improvement was detected. The totalamount of ABTS to be oxidized during thermal degradation by CLEAs and free laccasewas calculated and the insolubilized enzymes were reported to oxidize more substratethan free laccase. The formation conditions were analyzed by response surfacemethodology in order to determine an optimal environment for the production ofefficient laccase-based CLEAs using chitosan as the cross-linking agent. After 24hours of formation at pH 3 and at 4°C without agitation, theCLEAs exhibit the best specific activity