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Enzyme Research
Volume 2011 (2011), Article ID 376015, 10 pages
Research Article

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

1Environmental Engineering Laboratory, Department of Civil Engineering, University of Sherbrooke, 2 500 Boulevard de l'Université, Sherbrooke, QC, Canada J1K 2R1
2Department of Chemical and Biotechnological Engineering, University of Sherbrooke, 2 500 Boulevard de l'Université, Sherbrooke, QC, Canada J1K 2R1
3Étienne-Le Bel Centre de Recherche Clinique, Centre Hospitalier Universitaire de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4

Received 15 April 2011; Accepted 18 May 2011

Academic Editor: J. Guisan

Copyright © 2011 Alexandre Arsenault et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Laccase from Coriolopsis Polyzona was insolubilized as cross-linked enzyme aggregates (CLEAs) for the first time with chitosan as the cross-linking agent. Concentrations between 0.01 and 1.867 g/L of chitosan were used and between 0.05 and 600 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The laccase was precipitated using ammonium sulphate and cross-linked simultaneously. Specific activity and thermal stability of these biocatalysts were measured. Activities of up to 737 U/g were obtained when 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was used as a substrate. Moreover, the stability of these biocatalysts was improved with regards to thermal degradation compared to free laccase when exposed to denaturing conditions of high temperature and low pH. The CLEAs stability against chemical denaturants was also tested but no significant improvement was detected. The total amount of ABTS to be oxidized during thermal degradation by CLEAs and free laccase was calculated and the insolubilized enzymes were reported to oxidize more substrate than free laccase. The formation conditions were analyzed by response surface methodology in order to determine an optimal environment for the production of efficient laccase-based CLEAs using chitosan as the cross-linking agent. After 24 hours of formation at pH 3 and at 4°C without agitation, the CLEAs exhibit the best specific activity.