CHARACTERIZATION OF CELLULOLYTIC ACTIVITIES IN COMMERCIAL TRICHODERMA-REESEI PREPARATIONS - AN APPROACH USING SMALL, CHROMOGENIC SUBSTRATES

A comparison of the cellulolytic activities found in three commercial Trichoderma reesei preparations is presented. Classical substrates such as native or processed cellulose and substituted (soluble) derivatives (e.g. carboxymethylcellulose) are poorly defined. Chromogenic (fluorogenic) 4-methylumbelliferyl beta-glycosides can advantageously be applied to provide clear differentiation of the three main cellulolytic components of the complex from Trichoderma reesei, after separation by isoelectric focusing. Cellobiohydrolase I (CBH I) releases the fluorescent phenol from the lactoside (MULAC) and from the cellobioside (MUC). Endoglucanase I (EG I) liberates the phenol from the same substrates but the enzymes can be differentiated by specific inhibition with cellobiose (suppressing selectively the cellobiohydrolase activity). Endoglucanases III (EG III) is the only enzyme present in the complex capable of catalysing the hydrolysis of the cellotrioside (MUG)3 at the heterosidic bond, yielding the fluorescent 4-methylumbelliferone. The corresponding 2-chloro-4-nitrophenol glycosides offer an attractive alternative to classical reductometric methods. The substrates are sufficiently stable (pH 4-8; 60-degrees-C) and the favourable absorption characteristics of the phenol (pK 5.5, absorption coefficient at 405 nm 8750 M-1 cm-1, pH 5.6) allow sensitive, continuous and quantitative assays. Using these methods, it was found that Novo (Denmark) cellulase is particularly poor in beta-glucosidase activity, an IFP (France) preparation is maximally active on the classical substrates in contrast to the Genencor (USA) cellulase with high activity against the small, chromophoric substrates.