BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Homologous cloning, expression, and characterisation of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye Etienne Dubé & François Shareck & Yves Hurtubise & Claude Daneault & Marc Beauregard Received: 6 December 2007 / Revised: 13 March 2008 / Accepted: 28 March 2008 / Published online: 24 April 2008 # Springer-Verlag 2008 Abstract The lack of a commercially available robust and inexpensive laccase is a major barrier to the widespread application of this enzyme in various indus- trial sectors. By using an efficient system developed in Streptomyces lividans, we have produced by homologous expression 350 mg L -1 of a bacterial laccase with a high purity and without any extensive purification. This is the highest production yield reported in the literature for a bacterial laccase. The secreted enzyme achieved oxidation under a wide pH range depending on the substrate: 4.0 for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and 9.0 for 2,6-dimethoxyphenol. Furthermore, this bacterial laccase was found to be quite resistant under various conditions. It withstands pH from 3.0 to 9.0, shows a great thermostability at 70°C and was highly resistant toward conventional inhibitors. For instance, while the laccase of Trametes versicolor was completely inhibited by 1 mM NaN 3 , the laccase of Streptomyces coelicolor was fully active under the same conditions. To assess application potential of this laccase, we have investigated its ability to decolourise Indigo carmine. This enzyme was able to rapidly decolou- rise the dye in the presence of syringaldehyde as a redox mediator. Keywords Laccase . Streptomyces coelicolor . Homologous cloning . Expression . Characterisation . Dye decolourisation . Textile Introduction The first laccase studied was from Rhus vernicifera in 1883, a Japanese lacquer tree, from which the designation laccase was derived (Yoshida 1883). Laccases are defined in the Enzyme Commission nomenclature as oxidoreduc- tases acting on diphenols and related substances using molecular oxygen as acceptor (EC 1.10.3.2). They are multicopper proteins found mostly in plants and fungi but also in some bacteria (Alexandre and Zhulin 2000; Claus 2004; Sharma et al. 2007). The protein structure acts as an intricate ligand for the catalytically active coppers, provid- ing them with a coordination sphere where changes between the reduction states are thermodynamically possi- ble. They contain at least one type-1 copper, which is associated to the oxidation site, and typically harbour at least three additional coppers: one type-2 and two type-3 coppers arranged in a trinuclear cluster. This cluster is associated to the site where reduction of molecular oxygen occurs (Bertrand et al. 2002; Hakulinen et al. 2002; Piontek et al. 2002; Enguita et al. 2003). Laccases are of particular interest with regard to various commercial applications because of their ability to oxidise a wide range of relevant substrates. Thus, research is being carried out in various fields of interest: textile, pulp and paper, food and cosmetics industries, as well as in bioremediation, biosensor, biofuel and organic synthesis applications (Mayer and Staples 2002; Burton 2003; Kenealy and Jeffries 2003; Claus 2004; Rodriguez Couto et al. 2006; Minussi et al. 2007). Few commercialised Appl Microbiol Biotechnol (2008) 79:597–603 DOI 10.1007/s00253-008-1475-5 E. Dubé : Y. Hurtubise : C. Daneault : M. Beauregard (*) Département de chimie-biologie, Université du Québec à Trois-Rivières, 3351 Des Forges, C.P. 500, Trois-Rivières, Quebec, Canada, G9A 5H7 e-mail: marc.beauregard@uqtr.ca F. Shareck INRS-Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Ville de Laval, Quebec, Canada, H7V 1B7