Preparation of mutants of Trichoderma reesei with enhanced cellulase production

Abstract

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-fl-glucanase and ,B-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-,8-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulaseproducing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.

Enrichment technique for the selection of catabolite repression-resistant mutants of Trichoderma as producers of cellulase

“…Therefore, it appears that the production of cellulases by CDR-29 is only partially derepressed since the onset of cellulase production by this strain was delayed until the glucose concentration in the medium decreased under the certain level. Similar properties were described by Montenecourt and Eveleigh for their mutant RUT-NG14 [5].…”

“…To overcome these problems cellulase mutants which are resistant towards catabolite repression or mutants with constitutive synthesis of ceUulases have been sought [5][6][7][8][9].…”

“…The screening procedure consists in the growth of mutagenized conidia on agar plates containing phosphoric acid swollen cellulose (Walseth) plus 5% glycerol as the catabolite repressor [5,6]. The mutants resistant towards catabolite repression produce on the agar plates clearing zones of dissolved cellulose, also in the presence of glycerol.…”

Cloning, characterization and heterologous expression of the firstPenicillium echinulatumcellulase gene

“…This fact has led to the development of genetic approaches for obtaining new lineages of cellulolytic organisms with improved enzymatic production (Zhang et al 2006). Such programmes have been based on classical mutagenesis and selection (Montenecourt and Eveleigh 1977;Brown et al 1987;Dillon et al 1992Dillon et al , 2006Gadgil et al 1995;Anwar et al 1996;Chand et al 2005), protoplast fusion , as well as on the production of recombinant strains (Barnett et al 1991;Yoo and Pack 1992;Miettinen-Oinonen and Suominen 2002;Haakana et al 2004). In this genetic improvement scenario, a mutant strain of the cellulolytic fungus Penicillium echinulatum has been identified as a potential candidate for cellulases overproduction, becuase its secreting capacity is almost equivalent to the best fungal strains reported to date in the literature (Dillon et al 1992(Dillon et al , 2006.…”

Production of amylase by Aspergillus fumigatus utilizing α-methyl-?-glycoside, a synthetic analogue of maltose, as substrate

“…It was deposited in the Fundac°a ìo Tropical de Pesquisas e Tecnologia Andre è Tosello Collection, Campinas, Brazil and it was routinely maintained on potato dextrose agar at 30³C. The cultures were developed in 250-ml Erlenmeyer £asks containing 50 ml of Vogel salts [6] and 1% di¡erent carbon source at 30³C under stationary conditions. Flasks were harvested at periodic intervals, the contents ¢ltered through tarred Whatman #1 ¢lter paper and the mycelia dried to constant weight at 60³C for biomass determination.…”