Appl Microbiol Biotechnol (2006) 70: 740–746 DOI 10.1007/s00253-005-0122-7 APPLIED MICROBIAL AND CELL PHYSIOLOGY Aldo J. P. Dillon . Cinthia Zorgi . Marli Camassola . João Antônio P. Henriques Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities Received: 23 May 2005 / Revised: 20 July 2005 / Accepted: 24 July 2005 / Published online: 20 August 2005 # Springer-Verlag 2005 Abstract Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies show- ing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml -1 , measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of β-glucosidase per gram of wheat bran. Introduction Many factors have influenced the current interest in en- zymes that degrade, modify or transform cellulose; the main reasons for this interest are the investigation of the mechanisms of action of cellulases with the potential to hydrolyze lignocellulosic materials and the large potential commercial market for cellulases in the production of glucose syrups and fuel alcohol (Himmel et al. 1999). These interests have provided incentives for the develop- ment of genetic improvement programs to obtain news strains of cellulolytic organisms; such programs are based on mutagenesis and selection (Anwar et al. 1996; Brown et al. 1987; Chand et al. 2005; Dillon et al. 1992; Farkas et al. 1981; Gadgil et al. 1995), genetic recombination (Joseph et al. 1986; Manczinger and Ferenczy 1985; Meza et al. 1995) and the production of genetic transformants (Barnett et al. 1991; Miettinen-Oinonen and Suominen 2002; Yoo and Pack 1992). Concurrently, new and more effective fermentation processes have been developed (Sternberg and Dorval 1979; Tangnu et al. 1981). Understanding of the control systems involved in the expression of genes coding for cellulases is important in the development of genetic improvement programs for cellu- lase-producing organisms. Information is already available for Trichoderma reesei on basal cellulose-induced cellulase synthesis and the upstream region of the cellobiohydrolase I promoter gene cbh1 (Henrique-Silva et al. 1996; Torigoi et al. 1996), as well as on the feedback control exerted by the Trichoderma glucose repressor gene cre1 (Ilmén et al. 1996, 1997) and ace1 (Aro et al. 2003; Saloheimo et al. 2000), the regulatory transcriptional activating gene ace2. which is involved in the induction of cellulases and xylanase genes in T. reesei (Aro et al. 2001). Therefore, the generation of site-specific or random mutations in the region of the cbh1 promoter or in other cellulase gene promoters or in regions linked to the control of genes cre1, ace1 and ace2 seems to be a way to gain variant fungi with increased cellulase secretion. The most effective random mutation methods for ob- taining variants with enhanced cellulase production are those combining mutagenesis and enrichment, one of which is the use of the anti-metabolite 2-deoxyglucose (2DOG), which, when incorporated into a suitable medium contain- ing cellulose, causes catabolite repression of nonmutant strains, allowing only mutated strains resistant to this form of suppression to form colonies (Anwar et al. 1996; Farkas et al. 1981; Suzuki et al. 1996). In previous work, we isolated a strain of Penicillium echinulatum from the furniture beetle Anobium punctatum (De Geer) and used mutagenesis and selection to obtain mutant strains of P. echinulatum having cellulase filter paper activity (FPA) greater than 1.3 IU ml -1 (Dillon et al. 1992). In this paper, we describe a two-step methodology to create new P. echinulatum variants by using 2DOG in liquid medium followed by selection for increased cellu- lose hydrolysis on solid medium. A. J. P. Dillon (*) . C. Zorgi . M. Camassola . J. A. P. Henriques Institute of Biotechnology, University of Caxias do Sul, 1130 Francisco Getúlio Vargas Street, 95070-560 Caxias do Sul, RS, Brazil e-mail: ajpdillo@ucs.br