Purification and biochemical characterization of two xylanases produced by Aspergillus caespitosus and their potential for kraft pulp bleaching V.C. Sandrim a , A.C.S. Rizzatti b , H.F. Terenzi b , J.A. Jorge b , A.M.F. Milagres c , M.L.T.M. Polizeli b, * a Instituto de Quı ´mica, Universidade Estadual Paulista ‘Ju ´lio de Mesquita Filho’, Araraquara, SP, Brasil b Faculdade de Filosofia, Cie ˆncias e Letras de Ribeira ˜o Preto, Departamento de Biologia, Universidade de Sa ˜o Paulo, Avenida Bandeirantes, 3900 Bairro Monte Alegre, 14040-901 Ribeira ˜o Preto, SP, Brasil c Faculdade de Engenharia Quı ´mica de Lorena, Departamento de Biotecnologia, Lorena, SP, Brasil Received 7 November 2003; accepted 19 June 2004 Abstract Two extracellular xylanases produced by the thermotolerant fungus Aspergillus caespitosus grown in sugar cane bagasse were purified and characterized. Estimated molecular masses were 26.3 and 27 kDa (xyl I); 7.7 and 17.7 kDa (xyl II) for gel filtration and SDS-PAGE, respectively. Optimal temperature for both xylanases was 50–55 8C. Optimal pH was 6.5–7.0 for xyl I, and 5.5–6.5 for xyl II. The thermostability (T half ) at 55 8C was 27.3 min (xyl I) and >90 min (xyl II). Xylanase activity was inhibited by several ions. b-mercaptoethanol activated 59 and 102% xyl I and xyl II activities, respectively. These enzymes preferentially hydrolyzed birchwood xylan, and the K m and V max values were 2.5 mg/ml and 1679 U/mg protein (xyl I), and 3.9 mg/ml and 113 U/mg protein (xyl II). The action of both xylanases mainly that of xyl II, on kraft pulp reduced kappa number and increased pulp viscosity. # 2004 Elsevier Ltd. All rights reserved. Keywords: Aspergillus caespitosus; Xylanases; Pulp bleaching 1. Introduction Lignocellulose is the most abundant renewable biomass available in our planet. It comprises three major groups: cellulose, hemicellulose and lignin [1]. Xylan, the principal component of hemicellulose, is formed by a backbone of b- 1,4-linked-D-xylopyranosyl residues, and different substi- tute groups in the side chain. Hence, for complete degrada- tion of these polysaccharides an enzymatic complex including xylanases (1,4-b-D-xylan xylanohydrolase, EC 3.2.1.8) and b-xylosidases (1,4-b-D-xylan xylohydrolase, EC 3.2.1.37) which are responsible for the hydrolysis of the main chain is required. Xylanases cleave internal xylosidic linkages producing xylooligosaccharides, while b-xylosidases hydrolyze oligosaccharides releasing xylose [2]. The interest in xylanases has been raised markedly in the recent years, mainly because of their use in the pulp and paper industry, particularly in the bleaching process [3–5]. For such biotechnological applications xylanases are required to be stable at elevated temperatures, to be active at alkaline pH, and to be devoid of cellulase activity. Fungal xylanases from Aspergillus sp. and other microorganisms have been intensively studied [6–8]. The aim of this study was to purify and to characterize biochemically two extracellular xylanases produced by an isolate of Aspergillus caespitosus grown on sugar cane bagasse. Until now xylanases from this fungal species have not been reported in the literature. The effect of these two purified xylanases in pulp and paper bleaching was also investigated. www.elsevier.com/locate/procbio Process Biochemistry 40 (2005) 1823–1828 * Corresponding author Tel.: +55-16-602-3812; fax: +55-16-633-1758. E-mail address: polizeli@ffclrp.usp.br (M.L.T.M. Polizeli). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.06.061