Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects

Abstract

The activities of six purified Thermomonospora fusca cellulases and Trichoderma reesei CBHl and CBHll were determined on filter paper, swollen cellulose, and CMC. A simple method t o measure the soluble and insoluble reducing sugar products from the hydrolysis of filter paper was found t o effectively distinguish between exocellulases and endocellulases. Endocellulases produced 34% t o 50% insoluble reducing sugar and exocellulases produced less than 8% insoluble reducing sugar. The ability of a wide variety of mixtures of these cellulases t o digest 5.2% of a filter paper disc in 16 h was measured quantitatively. The specific activities of the mixtures varied from 0.41 to 16.31 pmol cellobiose per minute per micromole enzyme. The degree of synergism ranged from 0.4 t o 7.8. T. reesei CBHll and T. fusca E 3 were found to be functionally equivalent in mixtures. The catalytic domains (cd) of T. fusca endocellulases E2 and E 5 were purified and found to retain 93% and 100% of their CMC activity, respectively, but neither cd protein could digest filter paper to 5.2%. When E2cd and E5cd were substituted in synergistic mixtures for the native proteins, the mixtures containing E2cd retained 60%, and those containing E5cd retained 94% of the original activity. Addition of a P-glucosidase was found to double the activity of the best synergistic mixture. Addition of CBHl to T. fusca crude cellulase increased its activity on filter paper 1.7-fold. 0 1993 John Wiley & Sons, Inc.

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…Furthermore, the addition of CBMs in trans to the cognate catalytic module has led to a modest potentiation (0.2-to 1.5-fold) in catalytic activity against insoluble substrates (Din et al, 1994;Moser et al, 2008). Cellulases, typically endoglucanases, however, are often 3 orders of magnitude more active against soluble forms of cellulose than the crystalline polysaccharide (Durrant et al, 1991;Irwin et al, 1993). Thus, CBMs acting in trans have only a minor influence on the access problem, although this might reflect the dissociation of the targeting and (possible) disrupting function of these modules.…”

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

“…Cellulase enzymes produced by T. reesei have been shown to act synergistically to depolymerize cellulose in biomass with Cel7A playing the primary role of de-crystallizing and hydrolyzing cellulose microfibrils to its monomeric unit, cellobiose (Henrissat et al, 1985). T. reesei Cel7A has been shown to be highly effective on recalcitrant cellulose when acting alone, while also significantly enhancing the overall activity of cellulase mixtures (Irwin et al, 1993). The importance of the Cel7A enzyme in the cellulase system suggests that improvements in overall cellulase activity can be achieved by improving its access to cellulose.…”

An optimized microplate assay system for quantitative evaluation of plant cell wall–degrading enzyme activity of fungal culture extracts

“…The most effective means of lignocellulosic hydrolysis uses a cocktail of different cellulolytic enzymes, but conversion is still not optimal (Berlin et al, 2007;Cherry and Fidantsef, 2003;Irwin et al, 1993;Jeoh et al, 2002;Kabel et al, 2006). There is a need to develop more effective cocktails with a range of properties complementary to current cellulase systems, at a significantly reduced cost.…”