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INTRODUCTION The use of enzymes to treat endocrine disrupting chemicals (EDCs) and other emerging pollutants in wastewater is extremely attractive but developers must resolve many technical and economic issues. These include enzyme sensitivity to environmental conditions, the difficulty of retaining enzymes in practical reactors the relatively low concentration of EDC in wastewater, and the relatively high cost of enzymes for use in wastewater treatment systems. Our studies seek to develop methodologies for treating EDCs in wastewater using laccases in practical reactor systems Insolubilization of these biocatalysts as cross-linked enzyme aggregates (CLEAs) has been demonstrated to be quite promising in enhancing their stability, reusability and the possibility of continuous utilization in bioreactors (Ahuja et al. 2004). CLEAs of laccase secreted by the white rot fungus (WRF) Coriolopsis polyzona have been prepared by Cabana et al. (2007) using glutaraldehyde (GLU) as the cross-linking agent. These CLEAs have shown high enzyme activity and higher stability than free laccase against physical, chemical and biological denaturants and good kinetics of reaction. These biocatalysts have been successfully used for the continuous treatment of water contaminated by the endocrine disrupting chemicals bisphenol A, nonylphenol and triclosan (Cabana et al., 2009). The first objective of this study was to produce CLEAs of laccase and combinations of different laccases (Combi-CLEAs) by using chitosan as the cross-linking agent. The second objective was to determine the effects of the conditions of formation (pH, temperature, reaction time and shaking speed) on the characteristics of the CLEAs produced by this new approach. MATERIALS AND METHODS Trametes versicolor laccase (TvL) was purchased from Sigma-Aldrich (St-Louis, MO) and a bacterial laccase (MGL) was provided by MetGen Oy (Turku, Finland). Chitosan from crab shells (65% deacetylation), N-(3-dimethylaminopropyl)N'ethylcarbodiimide hydrochloride (EDC), 2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS), and 2,6 dimethoxyphenol (DMP) were purchased from either Fluka Biochemika or Sigma Aldrich . Enzymatic activity of free and insolubilized laccases were determined by spectrometrically monitoring the oxidation of ABTS at 420 nm, ε=36000 M-1cm-1 [3] for TvL and combi-CLEAs and the oxidation of DMP at 477 nm, ε=36000 M1cm-1 [4] for MGL and combi-CLEAs. One unit (U) of enzyme activity is defined as the amount of enzyme required to oxidize 1 µM of substrate (ABTS or DMP) per min. The following procedure was used to prepare the combi-CLEAs A total combined volume of 1 U mL-1 of TvL and MGL were precipitated on ammonium sulfate (500 g/L) for 30 min followed by an addition of solutions of chitosan (1 g/L) and EDC (50 mM) for aggregation and crosslinking. Acetate buffer at pH5 completed total solution to the desired volume (100 mL, usually) and stored at 4 °C for 48h. Subsequently, combi-CLEAs were extracted by centrifugation for 5 min at 10 000g and 4°C. The aliquots were then washed 3 times with deionized distilled water and used for experiments RESULTS AND DISCUSSION Graph1 shows the impact of pHs on the laccase activity of TvL CLEAs versus the Combi-CLEAs using ABTS as substrate at 20°C. One notes that both of these CLEAs have highest relative activity at pHs in the 3- 5 range. The CombiCLEAs have lower relative activity than the TvL CLEAs. Graph 2 shows the corresponding results using dimethoxyphenol (DMP) as substrate. The pH range has been extended and relative activities are better for pHs in the range of 8. Graph 3 and 4 shows the influence of temperature on the laccase activity at pH 4 and 8 using both ABTS and DMP as substrate. We are much more successful at obtaining high relative activities at ph 8 over a broad range of temperature (20 – 60°C) for DMP using Combi-CLEAs. Consequently, the Combi-CLEAs produced can be used over a wide temperature range without decrease in laccase activity. These novel laccasebased biocatalysts increase the potential of laccase utilization for wastewater treatment. Free enzymes (Trametes versicol and MetGen laccases) have been successfully insolubilized as combi-CLEAs. Combi-CLEAs synthesized exhibit optimal activity at 50°C with both ABTS and DMP. Activity retention appears high (50% to higher) with temperature variation for the biocatalyst except with ABTS at 70°C (>80% activity loss), which implies a great thermostability Similarly to the free enzymes, insolubilized fungal enzyme (TvL) is more oxidative in acidic pH than the bacterial enzyme (MGL) whereas the bacterial enzyme exhibit greater oxidation than the fungal enzyme in alkaline pH.· Acknowledgements: The authors are thankful to Antoine Mialon, PhD from MetGen Oy, Turku, Finland (http://www.metgen.fi) for providing the MetGen Laccase (MGL) enzymes.