Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Enhancing activity of cellulases is crucial for economically-viable lignocellulose conversion. We propose exploiting intermodular cooperativity to boost the activity of cellulosomal cellulases, including both multimodular cellulases with single catalytic modules and multifunctional cellulases containing two or more catalytic modules each. Three types of large “linkers”, categorized in terms of amino-acid composition and sequence as “proline/threonine-rich”, “proline/threonine-rich with additional glycine-enrichment”, and “generic (not enriched in any specific residues)” have been used in construction of such cellulases. Two chimeras of C. thermocellum CbhA were designed and constructed by replacement of its two consecutive X1 domains by each of the other two types of linkers; one of these linker-substituted CbhA constructs showed significant enhancement of its activity on crystalline cellulose relative to that of wild- type. Nine artificial multifunctional cellulases were constructed, so that all had a truncated C. thermocellum CbhA at the N-terminal, connected to each of three endoglucanase domains by each of three different linker segments, for a systematic 3x3 matrix testing linker-catalytic-domain interactions. Some of these multifunctional cellulases showed substantially higher activity than that of simple mixtures of their components. In order to further understand the effect of linkers on activity of multifunctional cellulases, four multifunctional cellulases were constructed with and without “linker” segments; activity of the former was much higher that that of the latter. All these results demonstrate that exploiting inter-modular (intra-peptide) cooperation is a promising approach to enhancing the cellulase capability of individual peptides.