RESEARCH PAPER 1 Trametes Hirsuta, an Excellent White rot Fungus for Laccase Production Rakesh U Thakare*, Arun B Ingle # , Bharat J Wadher*, Kunal Roychaudhury # * P.G. Department of Microbiology, RTM. Nagpur University, Nagpur, India # Department of Microbiology, S.K. Porwal College, Kamptee, Nagpur, India rakesh_rut@rediffmail.com Abstract Laccases are multi copper oxidases having wide substrate specificity mainly found in white-rot fungi, which are the only microorganisms able to degrade the whole wood components. As they are capable of degrading a wide variety of compounds they are commercially very significant. This project aims at studying the production optimization of laccase using different carbon sources. Agro-industrial residues, such as wheat bran, rice bran, and glucose were screened for laccase production, under aqueous-state fermentation conditions, by the white-rot fungus, Trametes hirsuta NCIM 1201. Glucose with guiuacol gave the highest activity, reaching about 1.2 U/ml within 10 days. Key words: laccase, aqueous-state fermentation, guiuacol, Trametes hirsuta NCIM 1201 INTRODUCTION Laccase is used industrially for pulp delignification, polycyclic aromatic hydrogen degradation, pesticide or insecticide degradation and organic synthesis. With microbial enzymes dominating world markets, more innovation and improvisation is needed to increase the efficiency of production at an economical rate. Laccase (E.C. 1.10.3.2) is the most common lignin modifying enzyme produced by the white-rot fungi belonging to the family Polyporaceae sensu lato (Pelaez et al. 1995). Among them, Trametes versicolor has extensively been used as the main experimental organism for laccase production studies. However, another fungus belonging to the same genus, Trametes hirsuta has also been described as a very promising candidate for the production of laccase (Vares and Hatakka., 1997). Moreover, laccase from T. hirsuta can efficiently degrade a wide variety of synthetic dyes (Abadulla et al. 2000 and Campos et al. 2001). This makes this biocatalyst very suitable for the treatment of wastewater from the textile industry. Most studies dealing with ligninolytic enzyme production by white-rot fungi have been carried out using liquid culture conditions (Rosales et al., 2007). The selection of a substrate for aqueous-state fermentation processes depends upon several factors mainly related with cost and availability and thus may involve screening of agro-industrial residues. Wheat bran has commonly been used for the cultivation of microorganisms in aqueous-state fermentation processes. In the present work, in addition to wheat bran, some other agro-industrial residues such as rice bran, glucose and glucose with guiuacol have been tested for laccase production by T. hirsuta under aqueous-state conditions. MATERIALS AND METHODS Microorganism: The white rot fungus, Trametes hirsuta NCIM-1201 was procured from National Center for Industrial Microorganisms, National Chemical Laboratory, Pune, India. Pure culture was maintained on potato dextrose agar. T. hirsuta NCIM 1201 was maintained and cultured in Potato Dextrose Agar (PDA) media and stored at 4°C. Fungal Staining: Lacto-phenol Cotton Blue was used for staining the fungal culture. Confirmation of laccase production: Laccases, which are extracellular secretion of white rot fungus, were able to oxidize different substrates such as guiuacol, syringoldazine, and non-phenolic compounds (Moorthi et al., 2007). The oxidase enzyme system of Trametes hirsuta NCIM 1201 was checked based on Trejo Hernandez et al. (2001). The laccase production media containing wheat bran flakes 4.5%, yeast extract 1.5%, glucose 1%, NH 4 Cl 0.25%, thiamine dichloride 0.05%, KH 2 PO 4 0.2%, MgSO 4 .7H 2 O 0.05%, CaCl 2 0.01%, KCl 0.05% and pH 5.0 was prepared and the guiuacol (50 μg/100 ml) was added as a substrate for laccase screening. In this study, a disc of 6 day-old culture was placed at the centre of the plate. The plates were incubated at the dark place and observed for reddish brown zone in the medium which will be formed as a result of laccase oxidative polymerization with guiuacol (Moorthi et al., 2007). Carbohydrate Sourcekk: The primary carbon source used was agricultural wastes which were used in place of Glucose and Guiuacol in a composite medium consisting of 0.1% KH 2 PO 4 , 0.05% MgSO 4 .7H 2 O, 0.02% NH 4 NO 3 , 0.001% CaCl 2 ,