Formation of enzyme polymer engineered structure for laccase and cross-linked laccase aggregates stabilization Thanina Hassani a , Sidy Ba b , Hubert Cabana a,c,⇑ a Environmental Engineering Laboratory, Department of Civil Engineering, University of Sherbrooke, 2500 Boulevard de l’Université, Sherbrooke, Quebec, Canada J1K 2R1 b Department of Chemical Engineering, University of Sherbrooke, 2500 Boulevard de l’Université, Sherbrooke, Quebec, Canada J1K 2R1 c Étienne-Le Bel Clinical Research Centre, Centre Hospitalier Universitaire de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4 highlights " The organic/inorganic network stabilized the enzyme versus harsh environments. " These structures did not limit the mass transfer of the substrate. " Applications of these biocatalysts are promising for bioprocesses. article info Article history: Received 20 May 2012 Received in revised form 9 October 2012 Accepted 12 October 2012 Available online 2 November 2012 Keywords: Laccase Cross-linked enzyme aggregates Enzyme polymer engineered structure Organic/inorganic surrounding network Chitosan abstract Laccase and laccase-based cross-linked enzyme aggregates (CLEAs) were stabilized through the formation of a surrounding polymeric network made of chitosan and 3-aminopropyltriethoxysilane. The thermoresistance of the resulting enzyme polymer engineered structures of laccase (EPES-lac) and CLEAs (EPES-CLEA) were more than 30 times higher than that of free laccase and CLEAs at pH 3 and 40 °C. The EPES showed higher residual activity than the unmodified biocatalysts against chaotropic salts (up to 10 times), EDTA (up to 5 times), methanol (up to 15 times) and acetone (up to 20 times). The Michaelis–Menten kinetic parameters revealed that the affinity for 2,2’-azino-bis-(3-ethylbenzothiazo- line-6-sulphonic acid) has doubled for the EPES-lac and EPES CLEA compared to their unmodified forms. The EPES-lac structures acted optimally at pH 4 and their activity was nearly temperature-independent, while the laccase activity of EPES-CLEA was optimal at pH 4 and 60 °C. Globally, the EPES have shown significantly improved properties which make them attractive candidate for the development of laccase-based applications. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Laccases (polyphenoloxidase, EC 1.10.3.2) mainly produced by plants, fungi, bacteria and insects, are able to catalyze the oxidation of many organic compounds such as methoxyphenols, phenols, o- and p-diphenols, aminophenols, polyphenols, poly- amines, molecules from lignin and some inorganic ions (Rodríguez Couto and Toca Herrera, 2006; Burton, 2003; Call and Mucke, 1997). These multicopper oxidases offer various applications including, cosmetics, food, textile and paper industry, wastewater treatment, and soil bioremediation (Call and Mucke, 1997; Selinheimo et al., 2006; Hou et al., 2004; Pointing, 2001). The use of free laccases in such applications is hampered by their relatively short lifetime and their instability under harsh environment (e.g., temperature, organic solvents, and salts) (Brady and Jordaan, 2009). In addition, the difficulty of retaining the free enzymes in a continuous flow bioreactor makes the use of laccases a costly alternative to conventional industrial processes (Osma et al., 2011). These drawbacks have impelled the search for strategies to en- hance the stability of the enzyme such as enzyme immobilization (with or without support), enzyme modification, genetic modifica- tion and medium engineering (Kim et al., 2006; Burton et al., 2002; Livage et al., 2001; Durán and Esposito, 2000; Koeller and Wong, 2001; Schmid et al., 2001). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.10.058 Abbreviations: EPES, enzyme polymer engineered structure; SENs, single enzyme nanoparticles; CA, carbonic anhydrase; APTES, 3-aminopropyltriethoxysi- lane; CLEAs, cross-linked enzyme aggregates; EDC, 1-ethyl-3-(3-dimethylamino- propyl)carbodiimide hydrochloride; ABTS, 2,2’-azino-bis-(3-ethylbenzothiazoline- 6-sulphonic acid); EPES-lac, enzyme-polymer engineered structure of laccase; EPES-CLEA, enzyme-polymer engineered structure of CLEAs. ⇑ Corresponding author at: Department of Civil Engineering, Environmental Engineering Laboratory, University of Sherbrooke, boulevard de l’Université, 2500 Sherbrooke (Qc), Canada J1K 2R1. Tel.: +1 819 821 8000x65457; fax: +1 819 821 7974. E-mail address: Hubert.Cabana@USherbrooke.ca (H. Cabana). Bioresource Technology 128 (2013) 640–645 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech