Folia Microbiol. 49 (1), 13–18 (2004) http://www.biomed.cas.cz/mbu/folia/ Purification and Characterization of Xylanases from Aspergillus giganteus M.B. FIALHO, E.C. CARMONA* Departamento de Bioquímica e Microbiologia, Instituto de Biociências, UNESP, 13 506-900, Rio Claro, São Paulo, Brazil fax 551 935 264 176 e-mail ecarmona@rc.unesp.br Received 14 April 2003 Revised version 11 August 2003 ABSTRACT. A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 ºC temperature optimum; optimum pH was 6.0–6.5 for xylanase I and 6.0 for xylanase II. At 50 ºC xylanase I exhibited higher thermostability than xylanase II. Hg 2+ , Cu 2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases. Hemicelluloses are noncellulose polysaccharides present in cell walls of terrestrial plants. Among them, xylan is the most common, representing up to 30 % of the wall dry mass. It is primarily located in the secondary cell wall, forming an interface between lignin and other polysaccharides (Biely 1985; Wong et al. 1988; Prade 1995). Most xylans are heteropolysaccharides formed by the main chain of 1,4--D-xylose monomers con- taining different substituents or ramifications. The most frequent substituents in the main xylan chain are acetyl, arabinosyl and glucuronosyl (Wong et al. 1988; Kulkarni 1999). Enzymic degradation of xylan in nature requires the action of two main enzymes produced mainly by fungi and bacteria, the endo-1,4--xylanases (1,4--D-xylan xylanohydrolases, EC 3.2.1.8) which hydro- lyze long-chain xylan and xyloolygosaccharides, thus releasing smaller oligosaccharides and xylobiose, which in their turn are broken down by the -xylosidases (1,4--D-xylan xylohydrolases, EC 3.2.1.37) which hydrolyze these products to xylose (Woodward 1984; Biely 1985; Augustín 2000; Kolarova and Augustín 2001; Zorec et al. 2001; Subramaniyan and Prema 2002). Xylan-degrading enzymes have been used for many biotechnological applications and, in some of them purified enzymes have been tested. The use of xylanolytic enzymes in the production of cellulosic pulp and its bleaching is important because it facilitates lignin removal from the pulp and reduces the consump- tion of chlorine chemicals as bleaching agents (Viikari 1994). Xylanase application also includes the bio- conversion of lignocellulosic material and agroindustrial residues into soluble sugars, treatment of juices, wine and beer production, oil extraction and improvement of animal feed digestibility (Woodward 1984; Biely 1985; Wong et al. 1988; Prade 1995; Bhat 2000; epeljnik et al. 2003). Among 879 strains isolated from Brazilian soil at the ecological station of Juréia-Itatins in the Mata Atlântica region, the strain of Aspergillus giganteus attracted attention as it exhibited the highest xylanolytic activity associated with a low cellulolytic activity, both being essential characteristics for some industrial use (Attili 1994). Coelho and Carmona (2003) studied the xylanolytic complex excreted by this fungus; they observed the highest xylanolytic activity on Vogel’s liquid medium containing oat spelts xylan as carbon source (pH 6.5–7.0) after 5-d stationary cultivation at 25 ºC. The aim of this work was to purify and bioche- mically characterize extracellular xylanases produced by A. giganteus after cultivation under optimized con- ditions. *Corresponding author.